Bone Morphogenetic Protein-2 And Bone Morphogenetic Protein-4 In The Treatment And Diagnosis Of Cancer

ABSTRACT

The present invention pertains to the use of BMP-2 and/or BMP-4 as 1) targets for cancer treatment therapies and 2) means to diagnose cancer. The therapeutic component of this invention involves administering to a patient a composition that inhibits bone morphogenetic protein-2 activity and/or bone morphogenetic protein-4 activity. Such inhibition may be accomplished by ligands or antibodies that bind to BMP-2 and/or BMP-4 or receptors for BMP-2 and BMP-4. It may also be achieved by preventing the processing of pro-BMP-2 and/or pro-BMP-4, or blocking transcription or replication of BMP-2 DNA and/or BMP-4 DNA or translation of BMP-2 mRNA and/or BMP-4 mRNA. The diagnostic component of the invention involves measuring the BMP-2 and/or BMP-4 level(s) in biological samples from both a patient and a non-cancerous subject and comparing those levels. Elevated levels of BMP-2 and/or BMP-4 in the patient compared to the subject indicate cancer.

[0001] This application claims the benefit of U.S. application Ser. No.10/044,716 (Langenfeld), filed Jan. 11, 2002. This application alsoincorporates by reference U.S. application Ser. No. 10/044,716.

FIELD OF USE

[0002] The present invention relates to the fields of molecular biology,immunology, and medicine and provides methods for the treatment anddiagnosis of cancer. Specifically, it relates to the use of bonemorphogenetic protein-2 (BMP-2) and bone morphogenetic protein-4 (BMP-4)as 1) targets for cancer treatment therapies and 2) means to diagnosecancer.

BACKGROUND OF THE INVENTION

[0003] Various publications or patents are referred to in parenthesesthroughout this application. Each of these publications or patents isincorporated by reference herein. Complete citations of the scientificpublications referred to in this section, the Background of theInvention, are set forth at the end of this section. All other citationsare set forth in the text.

[0004] Lung cancer is the leading cause of cancer deaths in the UnitedStates with over 150,000 people this year expected to die from thisdisease (1). Despite improvements in diagnosis and treatment, only 10%of lung cancer patients survive 5 years (1) with the majority ofpatients succumbing due to spread of the tumor to other parts of thebody. The genes that induce the invasion and metastasis of lung cancersare poorly understood. Applicant's experiments to identify genes thatregulate metastasis revealed that bone morphogenetic protein-2 (BMP-2)is overexpressed in human lung carcinomas. Subsequent experimentsrevealed that BMP-2 is also overexpressed in many other common humancancers. Applicant also found gene expression of BMP-4, a protein thatis highly homologous to BMP-2, in human lung cancer tumor samples.

[0005] BMP-2 and BMP-4 are powerful morphogenetic proteins that havebeen studied predominantly for their role in embryonic development andtheir ability to induce bone formation. The bone morphogenetic proteins(BMPs) are members of the transforming growth factor (TGF) superfamily,which are a phytogenetically conserved group of proteins (2). There are20 isotypes of the BMPs, with BMP-2 and BMP-4, which share 92% homology,placed in the same subclass based on their similar structures. (3, 4,5). BMP-2 and BMP-4 are secreted proteins that induce pluripotentialmesenchymal differentiation (6, 7) (8) and are required for the normalembryonic development of many organs including lung and bone (9, 10).BMP-2 and BMP-4 also have chemotactic properties capable of inducing themigration of normal vascular endothelial and mononuclear cells (12, 13).

[0006] The BMPs are synthesized as inactive variable length precursorproteins (14, 15). The precursor BMP-2 and BMP-4 proteins areproteolytically cleaved, producing mature C-terminal proteins of alittle more than 100 residues (4, 11, 14). BMP-2 and BMP-4 interact withthe same binding sites: mature BMP-2 and BMP-4 protein signaling ismediated by transmembrane serine/threonine kinases called type IA, IB,and type II receptors (4, 16-20). The receptor phosphorylatescytoplasmic targets, which includes the Smad family of proteins (21). Inaddition, the same molecules, including noggin, chordin, DAN, gremlin,and cerberus 1 homolog, inhibit both BMP-2 and BMP-4, thereby preventingtheir ability to bind to the receptors. (22-24)

[0007] While BMP expression has been noted in a few cancers, such assarcomas (25) and pancreatic cancer (26) and in cancer cell lines (27),the inhibition of BMP-2 activity and/or BMP-4 activity as a potentialcancer treatment has neither been mentioned nor studied in theliterature. To the contrary, several articles suggest that BMP-2 and/orBMP-4 have an inhibitory effect on cancer cell proliferation and may beuseful therapeutic agents to treat cancer. (28, 29, 30, 31)

[0008] Applicant has discovered that expression of bone morphogeneticprotein-2 (BMP-2) is linked to cancer invasion and growth and thatinhibiting BMP-2 activity reduces the size of cancerous tumors in nudemice and down regulates the expression of VEGF and sonic hedgehog inlung cancer cell lines. Applicant has also discovered BMP-4 geneexpression in human lung cancer tumors. As discussed above, BMP-2 andBMP-4 share very similar structures and nearly identical biologicalactivity. Thus, the present invention is directed toward using BMP-2and/or BMP-4 as targets for cancer treatment therapies and as a means todiagnose cancer. Specifically, the therapeutic component of thisinvention involves administering to a patient a composition thatinhibits bone morphogenetic protein-2 activity and/or bone morphogeneticprotein-4 activity. The diagnostic component of the invention involvesmeasuring the BMP-2 and/or BMP-4 level(s) in biological samples fromboth a patient and a non-cancerous subject and comparing those levels,with elevated levels indicating cancer in the patient.

[0009] References

[0010] 1. Thoracic Surgery, 1^(st) ed., vol. 1 (Churchill Livingstone,N.Y., Edinburgh, London, Melbourne, Tokyo) (1995).

[0011] 2. Warburton, D., et al. “The molecular basis of lungmorphogenesis” Mech Dev 92, 55-81 (2000).

[0012] 3. Celeste A J, et al. “Identification of transforming growthfactor beta family members present in bone-inductive protein purifiedfrom bovine bone” Proc Natl Acad Sci USA 87, 9843-9847 (1990).

[0013] 4. Leong, L. M., et al., “Bone Morphogenetic Protein-4” Int. J.Biochem. Cell Biol. 28, 1293-96 (1996).

[0014] 5. Zimmerman, L. B., et al. “The Spemann Organizer Signal NogginBinds and Inactivates Bone Morphogenetic Protein 4” Cell 86, 599-606(1996).

[0015] 6. Erickson, D. M., et al. “Recombinant bone morphogeneticprotein (BMP-2) regulates costochondral growth plate chondrocytes andinduces expression of BMP-2 and BMP-4 in a cell maturation-dependentmanner” J Orthop Res 15, 371-80 (1997).

[0016] 7. An, J., Rosen, et al. “Recombinant human bone morphogeneticprotein-2 induces a hematopoietic microenvironment in the rat thatsupports the growth of stem cells” Exp Hematol 24, 768-75 (1996).

[0017] 8. Abe, E., et al. “Essential requirement of BMPs-2/4 for bothosteoblast and osteoclast formation in murine bone marrow cultures fromadult mice: antagonism by noggin” J Bone Miner Res 15, 663-73 (2000).

[0018] 9. Vainio, S., et al. “Identification of BMP-4 as a signalmediating secondary induction between epithelial and mesenchymal tissuesduring early tooth development” Cell 75, 45-58 (1993).

[0019] 10. Weaver, M., et al. “BMP signaling regulates proximal-distaldifferentiation of endoderm in mouse lung development” Development 126,4005-15 (1999).

[0020] 11. Wozney, J. M., et al. “Novel regulators of bone formation:molecular clones and activities” Science 242, 1528-34 (1988).

[0021] 12. Willette, R. N., et al. “BMP-2 gene expression and effects onhuman vascular smooth muscle cells” J Vasc Res 36, 120-5 (1999).

[0022] 13. Cunningham, N. S., et al. “Osteogenin and recombinant bonemorphogenetic protein 2B are chemotactic for human monocytes andstimulate transforming growth factor beta 1 mRNA expression” Proc NatlAcad Sci USA 89, 11740-4 (1992).

[0023] 14. Cui, Y., et al. “BMP-4 is proteolytically activated by furinand/or PC6 during vertebrate embryonic development” Embo J 17, 4735-43(1998).

[0024] 15. Sugiura “Cloning and functional characterization of the5′-flanking region of the human bone morphogenetic protein-2 gene”Biochem J 3338, 443-440 (1999).

[0025] 16. Koenig, B. B., et al. “Characterization and Cloning of aReceptor for BMP-2 and BMP-4 form NIH 3T3 Cells” Molecular and CellularBiology 14, 5961-74 (1994).

[0026] 17. Liu, F., et al. “Human type II receptor for bonemorphogenetic proteins (BMPs): extension of the two-kinase receptormodel to the BMPs” Mol Cell Biol 15, 3479-86 (1995).

[0027] 18. Ikeda, T., et al. “Cloning of rat type I receptor cDNA forbone morphogenetic protein-2 and bone morphogenetic protein-4, and thelocalization compared with that of the ligands” Dev Dyn 206, 318-29(1996).

[0028] 19. ten Dijke, P., et al. “Activin receptor-like kinases: a novelsubclass of cell-surface receptors with predicted serine/threoninekinase activity” Oncogene 8, 2879-87 (1993).

[0029] 20. ten Dijke, P., et al. “Identification of type I receptors forosteogenic protein-1 and bone morphogenetic protein-4” J Biol Chem 269,16985-8 (1994).

[0030] 21. Sakou, T., et al. “Localization of Smads, the TGF-beta familyintracellular signaling components during endochondral ossification” JBone Miner Res 14, 1145-52 (1999).

[0031] 22. Piccolo, S., et al. “Dorsoventral patterning in Xenopus:Inhibition of ventral signals by direct binding of chordin to BMP-4”Cell 86, 589-98 (1996).

[0032] 23. Piek, E., et al. “Specificity, diversity, and regulation inTGF-β superfamily signaling” The FASEB Journal 13, 2105-24 (1999).

[0033] 24. McMahon, J. A., et al. “Noggin-mediated antagonism of BMPsignaling is required for growth and patterning of the neural tube andsomite” Genes & Development 12, 1438-52 (1998).

[0034] 25. Guo, W., et al. “Expression of bone morphogenetic proteinsand receptors in sarcomas” Clin. Orthop. 365: 175-83 (1999).

[0035] 26. Kleef, J., “Bone Morphogenetic Protein-2 exerts diverseeffects on cell growth in vitro and is expressed in human pancreaticcancer in vivo” Gastroenterology 116: 1202-1216 (1999).

[0036] 27. Hatakeyama, S., et al., “Expression of bone morphogeneticproteins of human neoplastic epithelial cells” Biochem Mol. Biol. Int.42(3): 497 (1997)

[0037] 28. Hjertner, O., et al., “Bone morphogenetic protein-4 inhibitsproliferation and induces apoptosis of multiple myeloma cells” Blood97(2): 516-22 (2001).

[0038] 29. Kawamura, C., et al., “Bone morphogenetic protein-2 inducesapoptosis in human myeloma cells with modulation of STAT3” Blood 96(6):2005-11 (2000).

[0039] 30. Soda, H. “Antiproliferative effects of recombinant human bonemorphogenetic protein-2 on human tumor colony-forming units” AnticancerDrugs 9(4): 327-31 (1998).

[0040] 31. Tada, A., et al., “Bone morphogenetic protein-2 suppressesthe transformed phenotype and restores actin microfilaments of humanlung carcinoma A549 cells” Oncol. Rep. 5(5): 137-40 (1998).

SUMMARY OF THE INVENTION

[0041] The present invention is related to Applicant's discovery thatbone morphogenetic protein-2 (BMP-2) is overexpressed in many commonhuman cancers and is linked to cancer invasion and growth. Further,inhibiting BMP-2 activity reduces the size of cancerous tumors in nudemice and down regulates the expression of VEGF and sonic hedgehog inlung cancer cell lines. In addition, gene expression of BMP-4 wasdetected in cancerous human lung tumors. As discussed above, BMP-4 ishighly homologous to BMP-2 and shares almost identical biologicalactivity with BMP-2. Thus, the present invention pertains to the use ofBMP-2 and/or BMP-4 as 1) targets for cancer treatment therapies and 2)means to diagnose cancer.

[0042] A primary aspect of the present invention is to provide a methodfor the treatment of cancer by administering to a patient atherapeutically effective amount of a BMP-2 and/or BMP-4 activityinhibitor. Some cancers that may be treated by this method arecarcinomas, including, but not limited to, lung cancer, bladder cancer,breast cancer, colon cancer, kidney cancer, ovarian cancer, thyroidcancer, endometrial cancer, omental cancer, testicular cancer, and livercancer. In a preferred embodiment of this invention the patient ishuman.

[0043] The BMP-2 inhibitor of this invention may be a polypeptide thatbinds specifically to bone morphogenetic protein-2, a polypeptide thatbinds specifically to a BMP-2 receptor, or an antibody that bindsspecifically to BMP-2. The BMP-2 inhibitor may also be an antisenseoligonucleotide that binds to a BMP-2 nucleic acid sequence or someportion thereof.

[0044] The BMP-4 inhibitor of this invention may be a polypeptide thatbinds specifically to bone morphogenetic protein-4, a polypeptide thatbinds specifically to a BMP-4 receptor, or an antibody that bindsspecifically to BMP-4. The BMP-4 inhibitor may also be an antisenseoligonucleotide that binds to a BMP-4 nucleic acid sequence or someportion thereof.

[0045] This invention features several particular polypeptides thatinhibit BMP-2 and/or BMP-4. Preferred embodiments of this inventionfeature known antagonists to BMP-2 and BMP-4, such as noggin, chordin,cerberus 1 homolog, gremlin, and DAN. Noggin is particularly preferred.Another aspect of this invention relates to the use of fragments ofnoggin, chordin, cerberus 1 homolog, gremlin, and DAN as BMP-2 and/orBMP-4 inhibitors.

[0046] Another embodiment of this invention provides a method fortreating cancer by administering to a patient a therapeuticallyeffective amount of an expression vector encoding a BMP-2 and/ or aBMP-4 inhibitor, such as a polypeptide that binds BMP-2 and/or BMP-4 orantisense oligonucleotides that bind to the nucleic acid for BMP-2and/or BMP-4. Another aspect of this invention includes the expressionvector described above in which the nucleic acid sequence that causesinhibition of BMP-2 and/or BMP-4 is operably linked to a selectivepromoter. One preferred selective promoter encompassed by this inventionis carcinoembryonic antigen promoter.

[0047] This invention also encompasses a kit that includes packagingmaterial, a BMP-2 activity inhibitor and/or a BMP-4 activity inhibitor,and instructions that indicate that the compounds can be used fortreating cancer in a patient. One type of cancer that may be treated iscarcinoma. Particular carcinomas encompassed by this invention are lungcancer, bladder cancer, breast cancer, colon cancer, kidney cancer,ovarian cancer, thyroid cancer, endometrial cancer, omental cancer,testicular cancer, and liver cancer.

[0048] The diagnostic component of this invention includes a method fordiagnosing cancer in a patient by obtaining a biological sample from apatient and measuring the level of BMP-2 and/or BMP-4 in the biologicalsample, with an elevated level or elevated levels of BMP-2 and/or BMP-4indicating cancer in the patient.

[0049] Any assay available to measure BMP-2 and/or BMP-4 levels isencompassed by this invention. Particularly preferred are immunoassays.Some examples of immunoassays included in this invention areEnzyme-Linked Immunosorbent Assay (ELISA), Western blot,immunoprecipitation, in situ immunohistochemistry, andimmunofluorescence. The Enzyme-Linked Immunosorbent Assay is mostparticularly preferred.

[0050] Another aspect of this invention is a method for the diagnosis ofcancer in a patient by detecting overexpression of BMP-2 and/or BMP-4 inthe patient by (i) quantifying in vivo or in vitro the presence of BMP-2and/or BMP-4 in a patient or a biological sample obtained from apatient, (ii) comparing the result obtained in step (i) to that of anormal, non-cancerous patient, and (iii) diagnosing for the presence ofcancer based on an increased level of BMP-2 and/or BMP-4 in step (ii)relative to a normal, non-cancerous patient.

BRIEF DESCRIPTION OF THE FIGURES

[0051]FIG. 1 illustrates representational difference analysis (RDA)subtraction. FIG. 1(a) shows amplification of CDNA prior to subtraction.Lane 1: IHBE cells; lane 2: lung carcinoma. FIG. 1(b) shows the distinctCDNA bands present after the second round of subtraction andamplification. FIG. 1(c) lists the proteins that were identified by aBLAST data base search after the DNA corresponding to each of the bandsshown in FIG. 1(b) was isolated and sequenced.

[0052]FIG. 2 is an ethidium-stained agarose gel showing the results ofRT-PCR performed on human lung cancer specimens. Lanes 1-4 contain theresults of the RT-PCR of various specimens, while lane 5 contains amarker.

[0053]FIG. 3 illustrates Western blots showing mature BMP-2overexpressed in lung cancer tissue specimens and lung cancer celllines. FIG. 3(a) is a representative Western blot showing overexpressionof BMP-2 in cancer tissue specimens. Lanes 1-5: normal lung tissue, lane6: SOAS osteosarcoma cell line, lanes 7-11: non-small lung cellcarcinomas. FIG. 3(b) is the corresponding actin immunoblot. FIG. 3(c)is a Western blot of non small cell lung carcinoma (NSCLC) subtypes.Lanes 1-4: normal lung tissue, lane 5: squamous carcinoma, lane 6:adenocarcinoma, lane 7: bronchoalveolar carcinoma, lane 8: large cellcarcinoma. FIG. 3(d) is the corresponding actin immunoblot. FIG. 3(e) isa BMP-2 immunoblot of lane 1: benign lung tumor, lane 2: mesthotheleoma,lane 3: normal lung tissue, lane 4: carcinoid tumor, lane 5: normallung, lane 6: NSCLC, lane 7: normal lung tissue, lane 8: NSCLC, lane 9:recombinant BMP-4. FIG. 3(f) is a BMP-4-probed Western blot with thesame lane contents as FIG. 3(e), except lane 9, which is recombinantBMP-4. FIG. 3(g) is the corresponding actin immunoblot.

[0054]FIG. 4(a) is a Western immunoblot of total cellular protein thatdemonstrates that normal and malignant human lung cell lines expressmature BMP-2 protein. Lanes (1) IHBE; (2) SOAS; (3) H7249; (4) A549. (b)Western blot of cell culture media shows lung cancer cell lines secretea BMP-2 precursor protein. Lanes (1) lung cancer tumor specimen; (2)A549 media; (3) H7249 media; (4) MHBE; (5), NBE media; (6) serum freemedia alone. (c) immunoblot of BMP type IA receptor. Lanes (1-3) normallung tissue specimens; (4) IHBE cells; (5) H7249 cells; (6) A549 cells;(7-9) lung cancer tissue specimens. (d) immunoblot of BMP type 1Breceptor. (1-3) normal lung tissue specimens; (4) IHBE cells; (5)H7249cells; (6) A549 cells; (7-9) lung cancer tissue specimens.

[0055]FIG. 5: 5(a) is an immunohistochemistry localizing BMP-2expression to the tumor cells. BMP-2 expression in a NSCLC demonstratingcytoplasmic staining of the tumor cells (arrowheads). The nuclei (n) ofthe tumor cells and the interstitium (I) are non-reactive; (b)Preabsorption of the BMP-2 antibody with recombinant human BMP-2 isnon-reactive with the tumor cells (arrows). Original magnification ×82.

[0056]FIG. 6(a) is a BMP-2 Western blot of human breast tumors andcorresponding normal tissue. Lane 1: NSCLC, lane 2-5: breast carcinomas,lane 6-8: normal breast tissue, lane 9: recombinant BMP-2. FIG. 6(b) isa BMP-2 Western blot of common human carcinomas and the correspondingnormal tissue. Lane 1: normal endometrium, lane 2: endometrialcarcinoma, lane 3: ovarian carcinoma, lane 4: normal colon, lane 5:colon carcinoma, lane 6: normal bladder, lane 7: bladder carcinoma.

[0057]FIG. 7(a) is a Western blot showing BMP-2 expression in metastatictumors. Lane 1: interstitial inflammatory lung disease, lane 2: normalomentum, lane 3: metastatic kidney tumor, lane 4: normal lymph node,lane 5: metastatic breast cancer, lane 6: metastatic kidney tumor, lane7: metastatic NSCLC, lane 8: omentum carcinoma. FIG. 7(b) is thecorresponding actin immunoblot. FIG. 7(c) is a BMPR IA Western blot,while FIG. 7(d) is a BMPR IB Western blot. The contents of the lanes onboth blots are the same: lane 1: normal kidney, lanes 2-3: normal lung,lane 4: metastatic kidney carcinoma, lane 5: metastatic breastcarcinoma, lane 6: metastatic NSCLC, lanes 7-9: NSCLC. FIG. 7(e) is BMPRIA Western blot and FIG. 7(f) is a BMPR IB Western blot of common humancarcinomas. Lane contents are the same on both blots: lane 1: normalkidney, lane 2: normal endometrium, lane 3: omentum, lane 4: normalcolon, lane 5: ovarian carcinoma, lane 6: kidney carcinoma, lane 7:endometrial carcinoma, lane 8: omenental Stumor, lane 9: coloncarcinoma.

[0058]FIG. 8 is a Western blot showing BMP-2 in serum samples from lungcancer patients. Lanes 1-2: serum samples, lane 3: recombinant BMP-2.

[0059]FIG. 9 shows that secreted BMP-2 precursor is proteolyticallycleaved by human leukocytes. Cell culture media from the A549 cellsincubated with leukocytes for 16 hours is probed with BMP-2 antibodyrecognizing its mature C-terminal end. FIG. 9(a) is the resultingWestern blot: lane 1: A549 lysate, lane 2: media without leukocytes,lanes 3-4: media with human leukocytes. FIG. 9(b) is the same immunoblothybridized with BMP-2 antibody recognizing its N-terminal end. FIG. 9(c)is a Western blot of leukocyte samples probed with anti-furin antibody.

[0060]FIG. 10 shows that BMP-2 treatment enhances formation of bloodvessels around a cancerous tumor. Each picture is of tissue from a nudemouse injected either with A549 cells or with A549 cells and BMP-2. Thepicture in the upper right shows tissue (including a tumor) from a nudemouse injected with A549 cells. Upper left: control. Upper right: mousetreated with BMP-2. Lower left: mouse treated with noggin.

[0061]FIG. 11 shows tissue (from nude mice injected with A549 cells andnude mice co-injected with A549 cells and BMP-2) stained with anti-CD 31antibody, which recognizes endothelial cells, viewed from under amicroscope. Left: control. Right: BMP-2 treated.

[0062]FIG. 12 shows that BMP-2 regulates sonic hedgehog expression. TheWestern blot on the left was probed with anti sonic hedgehog and showsan increase in sonic hedgehog expression as the amount of recombinantBMP-2 added to the A549 cell culture is increased. The Western blot onthe right was probed with anti sonic hedgehog and shows A549 cellculture media without added noggin (Lane 1) and cell culture media withadded noggin (Lane 2).

[0063]FIG. 13 shows that BMP-2 stimulates the migration of A549 andH7249 human lung cancer cell lines. 13(a): Recombinant human BMP-2, 1ng/ml, 10 ng/ml, 100 ng/ml, 500 ng/ml, or 1000 ng/ml was added to thelower well of the transwell chamber. Migrated cells counted usingfluorescent microscopy. 13(b) Noggin inhibits BMP-2 induced migration.Lane (1), media alone; (2) recombinant BMP2 (500 ng/ml); (3) noggin (10mg/mi) and recombinant BMP-2 (500 ng/ml). 13(c) H7249 cells migrated offcover slips towards Affi-Blue agarose beads containing recombinantBMP-2. 13(d) H7249 cells did not migrate off cover slips toward AffiBlueagarose beads containing dilution buffer. Similar results were foundusing the A549 cells. All the above experiments were repeated at least 3times. Data presented as mean+standard deviation. 13(e): Recombinanthuman BMP-2 stimulates the invasion of A549 or H7249 cells. RecombinantBMP-2, 1 ng/ml, 10 ng/ml, 100 ng/ml, 500 ng/ml, or 1000 ng/ml was addedto the lower wells of a Matrigel invasion chamber. Experiments wererepeated at least 3 times. Data presented as mean+5 standard deviation.

[0064]FIG. 14(a) show tumor growth after 19 days following thesubcutaneous co-injection of A549 lung cancer cells into nude mice withAffi-blue agarose beads coated with (1) 100 ug/ml of albumin, (2)recombinant human BMP-2, or (3) recombinant mouse noggin.

[0065]FIG. 15 show that noggin inhibits VEGF expression in the A549 lungcancer cell line. The Western blot was probed with anti-VEGF antibody.The lane labeled with a plus was cell culture media from culturestreated with noggin. The lane labeled with a minus was cell culturemedia from control cultures.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention is related to Applicant's discovery thatthe overexpression of bone morphogenetic protein-2 (BMP-2) is linked tocancer invasion and growth. BMP-2 is overexpressed in many common humancancers and regulates molecular pathways that are involved in thepromotion of cancer. Inhibiting BMP-2 activity reduces the size ofcancerous tumors in nude mice and down regulates the expression of VEGFand sonic hedgehog, which have been linked to cancer, in lung cancercell lines. In addition, BMP-4 gene expression was detected in humanlung cancer tumor specimens. BMP-4 is highly homologous to BMP-2, it isinhibited by the same molecules that inhibit BMP-2, and it binds thesame receptors as does BMP-2. See Piek, E., et al. “Specificity,diversity, and regulation in TGF-β superfamily signaling” The FASEBJournal 13, 2105-24 (1999); Leong, L. M., et al., “Bone MorphogeneticProtein-4” Int. J. Biochem. Cell Biol. 28, 1293-96 (1996); Zimmerman, L.B., et al. “The Spemann Organizer Signal Noggin Binds and InactivatesBone Morphogenetic Protein 4” Cell 86, 599-606 (1996); Piccolo, S., etal. “Dorsoventral patterning in Xenopus: Inhibition of ventral signalsby direct binding of chordin to BMP-4” Cell 86, 589-98 (1996). Thus, thepresent invention is directed toward using BMP-2 and/or BMP-4 as atarget for cancer treatment therapies and as a means to diagnose cancer.

[0067] The therapeutic component of this invention involvesadministering to a patient a composition that inhibits bonemorphogenetic protein-2 activity and/or bone morphogenetic protein-4activity. Such inhibition may be accomplished by ligands or antibodiesthat bind to BMP-2 and/or BMP-4 or receptors for BMP-2 and/or BMP-4. Itmay also be achieved by preventing the processing of pro-BMP-2 and/orpro-BMP-4, or blocking transcription or replication of BMP-2 DNA and/orBMP-4 DNA or translation of BMP-2 MRNA and/or BMP-4 MRNA. Delivery ofsuch compositions may be systemic or tissue-targeted.

[0068] The diagnostic component of the invention involves measuring theBMP-2 and/or BMP-4(s) level in biological samples from both a patientand a non-cancerous subject and comparing those levels. Elevated levelsof BMP-2 and/or BMP-4 in the patient compared to the subject indicatecancer.

[0069] Although specific embodiments of the present invention will nowbe described, it should be understood that such embodiments are examplesthat are merely illustrative of a small number of the many possiblespecific embodiments that can represent applications of the principlesof the present invention. Various modifications obvious to one skilledin the art to which the present invention pertains are within thespirit, scope and contemplation of the present invention as furtherdefined in the appended claims.

[0070] Definitions

[0071] A “bone morphogenetic protein-2 activity inhibitor” is acomposition that antagonizes the activity of the BMP-2 protein byspecifically binding to it or to BMP receptors, blocks the activation ofpro-BMP-2, or prevents the replication or transcription of the BMP-2gene or the translation of BMP-2 MRNA into protein.

[0072] A “bone morphogenetic protein-4 activity inhibitor” is acomposition that antagonizes the activity of the BMP-4 protein byspecifically binding to it or to BMP receptors, blocks the activation ofpro-BMP-4, or prevents the replication or transcription of the BMP-4gene or the translation of BMP-4 MRNA into protein.

[0073] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds. Polypeptide refers to both short chains, commonlyreferred to as peptides, oligopeptides or oligomers, and to longerchains, generally referred to as proteins. Polypeptides include aminoacid sequences modified either by natural processes, such asposttranslational processing, or by chemical modification techniquesthat are well known in the art. Such modifications are well described inbasic texts and in more detailed monographs, as well as in a voluminousresearch literature.

[0074] An “expression vector” is a recombinant vector that incorporatesthe desired gene and associated control sequences that promote and/orregulate expression of the gene. The desired gene is “operably linked”to such control sequences. The term “operably linked” means that theregulatory sequences necessary for expression of the coding sequence areplaced in an appropriate position in the expression vector relative tothe coding sequence so as to enable expression of the coding sequence.The preparation of such recombinant expression vectors as well as theuse of various control sequences is well known to those of skill in theart and described in many references. See, for example, Sambrook, J., etal., Molecular Cloning : A Laboratory Manual 2nd ed. (Cold SpringHarbor, N.Y., Cold Spring Harbor Laboratory) (1989).

[0075] A “selective promoter” refers to a promoter that is notindiscriminately expressed. Instead it is expressed only, for example,in certain tissues, certain tumors, in response to certain treatments,or in response to certain events in a cell. Such tissue-specific,tumor-selective, treatment-responsive, or tumor endothelium directedpromoters are described in Nettlebeck, D. M., et al., “Gene therapy:designer promoters for tumour targeting” Trends Genet 16(4); 174-81(2000).

[0076] An “expression vector vehicle”refers to an expression vectorpaired with a moiety that facilitates delivery of the expressionconstruct to cells in vivo. An expression vector may incorporate genesencoding the delivery moiety. One example of such an expression vectoris a viral vector.

[0077] The term “antibody” refers to polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of a Fab or other immunoglobulinexpression library.

[0078] “Polyclonal” refers to antibodies that are heterogeneouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, especially when using an entireprotein, or a larger section of the protein. The type of adjuvant usedwill depend on the hosts. Typical adjuvants include Fruend's, Fruend'scomplete, or oil-in-water emulsions. In these cases the entire proteinor portion thereof can serve as the antigen. When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate for use as the antigen.Commonly utilized conjugate proteins that are commercially available forsuch use include bovine serum albumin (BSA) and keyhole limpethemocyanin (KLH).

[0079] “Monoclonal antibodies” are substantially homogeneous populationsof antibodies to a particular antigen. They may be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. Such methods are well known to thoseof ordinary skill in the art and include general hybridoma methods ofKohler and Milstein, Nature (1975) 256: 495-497, the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today4:72 (1983) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc. (1985). Thebasic technique involves injecting a mouse, or other suitable animal,with an antigen. The animal is subsequently sacrificed and cells takenfrom its spleen are fused with myeloma cells. The result is a hybridcell, referred to as a hybridoma, that reproduces in vitro. Thepopulation of hybridomas are screened to isolate individual clones eachof which secrete a single antibody species to the antigen. Theindividual antibody species obtained in this way are each the product ofa single B cell from the immune animal generated in response to aspecific antigenic site recognized on the antigen. Kohler, G. andMilstein, C. Nature (London) 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976).

[0080] The term “antibody fragment” refers to a portion of an antibody,often the hyper variable region and portions of the surrounding heavyand light chains, that displays specific binding affinity for aparticular molecule. The term antibody fragment also includes singlechain antibodies.

[0081] An “antisense oligonucleotide” is an oligonucleotide thatspecifically hybridizes, under cellular conditions, with the cellularMRNA or genomic DNA encoding a BMP-2 protein and/or with the cellularmRNA or genomic DNA encoding a BMP-4 protein or some portion of suchcellular or genomic DNA, thereby inhibiting biosynthesis of the BMP-2and/or BMP-4 protein. The binding may be via conventional base paircomplementarity, or, in the case of binding to DNA duplexes, viaspecific interactions in the major groove of the double helix.

[0082] The term “effective amount” refers to the quantity of a compoundthat is sufficient to yield a desired therapeutic response without undueadverse side effects (such as toxicity, irritation, or allergicresponse) commensurate with a reasonable benefit/risk ratio when used inthe manner of this invention. The specific “effective amount” will,obviously, vary with such factors as the particular cancer beingtreated, the physical condition of the patient, the type of mammal beingtreated, the duration of the treatment, the nature of concurrent therapy(if any), and the specific formulations employed and the structure ofthe compounds or their derivatives.

[0083] A “patient” is a mammal suspected of having cancer. The patientis preferably human but may also be another mammal, such as a cat, dog,horse, cow, rat, or mouse.

[0084] A “biological sample” is a substance obtained from the patient'sbody. The particular “biological sample” selected will vary based on thecancer the patient is suspected of having and, accordingly, whichbiological sample is most likely to contain BMP-2 and/or BMP-4.

[0085] An “elevated level” means the level of bone morphogeneticprotein-2 and/or bone morphogenetic protein-4 that is greater than thelevel of analyte present in a particular biological sample of patientthat is not suffering from cancer.

[0086] A “carcinoma” is an epithelial cancer. Examples of carcinomas arebladder cancer, breast cancer, colon cancer, kidney cancer, lung cancer,ovarian cancer, thyroid cancer, endometrial cancer, omental cancer,testicular cancer, and liver cancer. The epithelium predominately linesducts and lining of organs or glands.

[0087] BMP-2 and/or BMP-4 as a Target in the Treatment of Cancer

[0088] The present invention is directed to the use of BMP-2 and/orBMP-4 as a target in the treatment of cancer. Amino acids #283-396 ofSEQ ID NO: 2 constitute the amino acid sequence of mature human BMP-2.Nucleotides #372-1514 of SEQ ID NO: 1 constitute the nucleotide codingsequence for human BMP-2. Amino acids #293-408 of SEQ ID NO: 18constitute the amino acid sequence of mature human BMP-4. Nucleotides#3166-10271 of SEQ ID NO: 17 constitute the gene for human BMP-4. Exon#3 (nucleotides #7791-8167) and exon #4 (nucleotides #9131-10271)encompass the nucleotide coding sequence and 5′ and 3′ flanking regionsfor human BMP-4. Given the experiments described above, the 92% homologybetween BMP-2 and BMP-4, and their binding of the same receptors andinhibitors, any composition that 1) specifically binds BMP-2 and/orBMP-4 or a receptor for BMP-2 and/or BMP-4, thereby antagonizing BMP-2and/or BMP-4 activity, 2) blocks the processing of pro-BMP-2and/proBMP-2, or 3) prevents the replication or transcription of BMP-2and/or BMP-4 DNA or the translation of BMP-2 and/or BMP-4 mRNA could beused as a therapy to treat cancer.

[0089] A compound that specifically binds to BMP-2 is any compound (suchas a polypeptide or an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of BMP-2.As one of ordinary skill in the art will appreciate, such “specific”binding compounds may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater than99% identity) with the amino acid sequence of BMP-2. Thus, a compoundthat specifically binds BMP-2 may also specifically bind BMP-4, to whichBMP-2 is 92% homologous, thereby inhibiting BMP-4 activity as well.

[0090] A compound that specifically binds to BMP-4 is any compound (suchas a polypeptide or an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of BMP-4.As one of ordinary skill in the art will appreciate, such “specific”binding compounds may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater than99% identity) with the amino acid sequence of BMP-4. Thus, a compoundthat specifically binds BMP-4 may also specifically bind BMP-2, therebyinhibiting its activity as well.

[0091] Similarly, a compound that specifically binds to a BMP receptoris any compound that has a binding affinity for any naturally occurringisoform, splice variant, or polymorphism of the BMP receptor. As one ofordinary skill in the art will appreciate, such “specific” bindingcompounds may also bind to other closely related proteins that exhibitsignificant homology (such as greater than 90% identity, more preferablygreater than 95% identity, and most preferably greater than 99%identity) with the amino acid sequence of a BMP receptor.

[0092] The present invention embodies polypeptides that specificallybind to BMP-2 and/or BMP-4 or that specifically bind to BMP receptors,thereby inhibiting BMP-2 and/or BMP-4 activity. Specific embodiments ofsuch polypeptides are described below.

[0093] The present invention encompasses known antagonists of BMP-2 andBMP-4 activity, including noggin (Brunet, L. J., et al., “Noggin,Cartilage Morphogenesis, and Joint Formation in the Mammalian Skeleton”Science 280(5368): 1455-7 (1998); U.S. Pat. No. 6,075,007, Economides,et al.), chordin (U.S. Pat. No. 5,896,056, LaVallie, et al.; Millet, C.,et al., “The human chordin gene encodes several differentially splicedvariants with distinct BMP opposing activities” Mech. Dev. 106(1-2):85-96 (2001)), gremlin (GenBank Accession No.

[0094] AF154054), cerberus 1 homolog (GenBank Accession No.NM_(—)005454), and DAN.

[0095] Recombinant mouse noggin from R & D Systems (Minneapolis, Minn.)was used in the inhibition experiments described in the Results sectionbelow. Mouse and human noggin share 98% homology. Therefore, thisinvention also relates to use of a polypeptide with the amino acidsequence of mature mouse noggin (amino acids #20-231 of SEQ ID NO: 6)and with the amino acid sequence of mature human noggin (amino acids#20-231 of SEQ ID No.: 4) as a BMP-2 activity inhibitor and a BMP-4activity inhibitor. The amino acid sequence for human chordin is SEQ IDNo: 8, for human gremlin is SEQ ID NO: 10, and for cerberus 1 homolog isSEQ ID NO: 12. The nucleotide coding sequence for human noggin is SEQ IDNO: 3, for mouse noggin is SEQ ID NO: 5, for human chordin isnucleotides #247-3114 of SEQ ID NO: 7, for human gremlin is nucleotides#130-684 of SEQ ID NO: 9, for human cerberus 1 homolog is SEQ ID NO: 11.

[0096] This invention also embodies polypeptide fragments of noggin,chordin, gremlin, cerberus 1 homolog, and DAN that bind BMP-2 and/orBMP-4, thereby inhibiting the activity of BMP-2 and/or BMP-4. Suchpolypeptides may be tested for inhibitory efficiency by culturing cellstransformed with progressively shorter portions of the nucleotidesequences encoding the above proteins, recovering and purifying from thevarious cultures the resulting polypeptide, and testing thosepolypeptides for their ability to inhibit BMP-2 activity and/or BMP-4activity.

[0097] This invention also includes genetically altered BMP receptorproteins that inhibit BMP-2 activity and/or BMP-4 activity. For example,altered BMP receptors that inhibit the binding effects of BMP-2 and/orBMP-4 are described in U.S. Pat. No. 6,291,206 (Wozney, et al.)

[0098] Also included by this invention are polypeptides that bind BMPreceptors without activating them. (Nickel, J., et al. “The CrystalStructure of the BMP-2:BMPR-IA Complex and the Generation of BMP-2Antagonists” The Journal of Bone & Joint Surgery 83-A, Supp.1, Part 1:7-14 (2001). Kirsch, T., et al. “BMP-2 antagonists emerge fromalterations in the low-affinity binding epitope for receptor BMPR-II”The EMBO Journal 19(13):3314-24 (2000)) Particularly preferred areligands that will bind BMP IB receptors, as aberrant expression of theBMP IB receptor in many human cancer specimens has been noted, asdiscussed in the Results section below. (Ide, H., et al., “Cloning ofhuman bone morphogenetic protein type IB receptor (BMPR-IB) and itsexpression in prostate cancer in comparison with other BMPRs” Oncogene13(11): 1377-82 (1997)). The coding sequence for BMP IB precursor isnucleotides #274-1782 of SEQ ID NO: 13. The amino acid sequence for BMPIB is amino acids #14-502 of SEQ ID NO 14.

[0099] This invention also encompasses expression vectors thatincorporate a nucleotide sequence encoding an inhibitor of BMP-2activity and/or BMP-4 activity operably linked to control sequences thatpromote and/or regulate expression of the nucleotide sequence. Thepreparation of such expression vectors as well as the use of variouscontrol sequences is well known to those of skill in the art and isdescribed in many references, such as Sambrook, et al. (1989).Expression vectors can be derived from bacterial plasmids, frombacteriophage, from transposon, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses and fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Promoterscan be prokaryotic, such as lac, lacz, T3, T7, gpt, lambda PR, PL, andtrp, or eukaryotic, such as CMV immediate early, HSV thymidine kinase,early and late SV40, LTR's from retrovirus, and mouse metallothionein-1.Selective promoters such as those described in Nettlebeck, D. M., etal., “Gene therapy: designer promoters for tumour targeting” TrendsGenet 16(4); 174-81 (2000) that are tissue-specific, tumor-selective,treatment-responsive, or tumor endothelium directed may also be used.For example, the promoter of the carcinoembryonic antigen (CEA) isexpressed on many breast, lung, and colorectal cancers.

[0100] For introduction of a gene that encodes a protein thatantagonizes BMP-2 activity and/or BMP-4 activity an expression vectorvehicle that will facilitate delivery of the desired gene to theaffected cells may be used. One way to facilitate delivery is by usingan expression vector derived from virus. Examples of viral vectors thathave been successfully used to deliver desired sequences to cells withhigh infection efficiency are adenoviral, retroviral, vaccinia viral,and adeno-associated viral vectors. Commonly used viral promoters forexpression vectors are derived from polyoma, cytomegalovirus,Adenovirus, and Simian Virus 40 (SV40). It is also possible to usepromoter or control sequences normally associated with the desired genesequence, if such control sequences are compatible with the host cellsystems.

[0101] Non-viral expression vector vehicles are also available. Forinstance, the expression vector could be associated with one or morelipids. As is known in the art of lipid-based gene delivery, suchnucleic acid-lipid complexes can be in a variety of different formsdepending generally on the nature of the lipid employed, the ratio ofnucleic acid to lipid and/or other possible components, and the methodby which the complex is formed. Examples of complexes include liposomesand micelles. Liposome-mediated gene transfer seems to have greatpotential for certain in vivo applications in animals. Studies haveshown that intravenously injected liposomes are taken up essentially inthe liver and the spleen, by the macrophages of the reticuloendothelialsystem. Using a catheter to introduce liposomes coupled to expressionvectors to particular cellular sites has also been described. (Nabel, E.G., et al., Science 249:1285-1288 (1990))

[0102] Another possible expression vector vehicle consists of a cellreceptor-specific ligand and a DNA-binding agent that would bind to theexpression vector. (Nishikawa, M. et al., Gene Therapy 7:548-55 (2000)).Such a vehicle could also comprise a cell receptor-specific ligand andthe nucleic acid-lipid complex described above. Nicolau, C. et al.,Methods Enzvmol 149: 157-76 (1987))

[0103] In addition, the present invention embodies antibodies thatspecifically bind BMP receptors or BMP-2 and/or BMP-4, therebyinhibiting BMP-2 activity and/or BMP-4 activity. When raising antibodiesto BMP-2, BMP-4, or BMP receptors, the entire protein (either theprecursor or the processed protein), or a portion thereof, may beutilized. Information usefuil in designing an antigen for the productionof antibodies to BMP-2 may be deduced by those of skill in the art byhomology analysis of SEQ ID NO: 2, especially amino acids #283-396 ofSEQ ID NO: 2. Information useful in designing an antigen for theproduction of antibodies to BMP-4 may be deduced by those of skill inthe art by homology analysis of SEQ ID NO: 18, especially amino acids#293-408 of SEQ ID NO: 18. Antibodies that recognize both BMP-2 andBMP-4 could be designed by one of skill in the art by analyzing theamino acid sequences of both proteins.

[0104] Recombinant human BMP-2 and BMP-4 proteins are commerciallyavailable from R & D Systems (Minneapolis, Minn.) and portions of theBMP-2 and BMP-4 proteins may be produced by a variety of methods. Inorder to raise antibodies to particular epitopes, peptides derived fromthe full BMP-2 or the full BMP-4 sequence may be used.Custom-synthesized peptides in the range of 10-20 amino acids areavailable from a multitude of vendors, and can be ordered conjugated toKLH or BSA. Alternatively, peptides in excess of 30 amino acids may besynthesized by solid-phase methods, or may be recombinantly produced ina recombinant protein production system. In order to ensure properprotein glycosylation and processing an animal cell system (e.g., Sf9 orother insect cells, CHO or other mammalian cells) is preferred.

[0105] Selection of antibodies which alter the activity of BMP-2 and/orBMP-4 may be accomplished in several ways. Antibodies that alter thebinding of BMP-2 and/or BMP-4 to a receptor may be detected by wellknown binding inhibition assays. For instance, according to standardtechniques, the binding of a labeled (e.g., flourescently orenzyme-labeled) antibody to BMP-2, which has been immobilized in amicrotiter well, is assayed for BMP-2 binding in both the presence andabsence of the appropriate receptor. The decrease in binding will beindicative of a competitive inhibitor relationship between the antibodyand the receptor. The same technique could be used with BMP-4. Inaddition, antibodies that are useful for altering the function of BMP-2and/or BMP-4 may be assayed in functional formats, such as the cellmigration assays described in the Results and Examples sections.

[0106] This invention also embodies compositions that prevent theprocessing of inactive BMP-2 and/or BMP-4 precursors. BMP precursors areproteolytically activated by proprotein convertases. For example,pro-BMP-2 is cleaved by furin convertase from human leukocytes. Inaddition, pro-BMP-4 is cleaved by furin and/ or PC6. See Cui, Y., et al.“BMP-4 is proteolytically activated by furin and/or PC6 duringvertebrate embryonic development” The EMBO Journal 17, 4735-43 (1998).Furin inhibitors are known. See, e.g., Cui, Y. et al.; Cameron, A., etal., “Polyarginines are potent furin inhibitors” J. Biol. Chem. 275:36741-49 (2000).

[0107] While the BMP-2 and BMP-4 inhibitors discussed above adverselyaffect BMP-2 activity and/or BMP-4 activity after these proteins areexpressed, it will be readily apparent to one of ordinary skill in theart that specific prevention of BMP-2 and/or BMP-4 biosynthesis willachieve the same goals as more direct inhibition of activity.Consequently, this invention also encompasses inhibition of BMP-2 and/orBMP-4 biosynthesis as a method for treating cancer. Such inhibition maybe achieved by selectively degrading mRNA encoding BMP-2 and/or mRNAencoding BMP-4 or by interfering with transcription or translation ofsuch mRNA. See Glavic, A., et al., “Xiro-1 controls mesoderm patterningby repressing BMP-4 expression in the Spemann organizer” Dev. Dyn.222(3): 368-376.

[0108] Inhibition of BMP-2 and/or BMP-4 biosynthesis to treat for cancercould also be achieved through antisense therapy. Antisense therapy isthe administration or in situ generation of oligonucleotides thatspecifically hybridize, under cellular conditions, with the cellularMRNA or genomic DNA encoding a BMP-2 or BMP-4 protein or some portion ofsuch cellular or genomic DNA, thereby inhibiting biosynthesis of theBMP-2 or BMP-4 protein. Antisense therapy refers generally to the rangeof techniques known by one of ordinary skill in the art, and includesany therapy that relies on specific binding to oligonucleotidesequences.

[0109] Delivery of an antisense oligonucleotide of the present inventioncan occur in a variety of ways. For example, antisense oligonucleotidescan be delivered as expression vectors that produces RNA which iscomplementary to at least a unique portion of the cellular mRNA encodingBMP-2 and/or the cellular mRNA encoding BMP-2. Such an expression vectorcould be delivered to cells by one of the expression vector vehiclesdescribed above. Alternatively, the antisense oligonucleotide could begenerated ex vivo as an oligonucleotide probe which, when introduced tothe cell, inhibits biosynthesis of BMP-2 and/or BMP-4 proteins byhybridizing with the MRNA or genomic sequences encoding BMP-2 and/orBMP-4. Such oligonucleotide probes could be modified oligonucleotidesthat are resistant to endogenous nucleases and therefore are stable invivo. General methods to construct oligomers useful in antisense therapyare known in the art. (Van der krol, et al., Biotechniques 6:958-976(1988); Stein, et al., Cancer Res. 48:2659-2668 (1988).

[0110] Dosage forms of the inhibitors of BMP-2 and/or BMP-4 of thisinvention include pharmaceutically acceptable carriers known to those ofordinary skill in the art. Pharmaceutically acceptable components arethose that are suitable for use with mammals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio. The carrier can be asolid or liquid and the type is generally chosen based on the type ofadministration being used. The active agent can be coadministered in theform of a tablet or capsule, as an agglomerated powder or in a liquidform. Examples of suitable solid carriers include lactose, sucrose,gelatin and agar. Capsule or tablets can be easily formulated and can bemade easy to swallow or chew; other solid forms include granules andbulk powders. Tablets may contain suitable binders, lubricants,diluents, disintegrating agents, coloring agents, flavoring agents,flow-inducing agents and melting agents. Examples of suitable liquiddosage forms include solutions or suspensions in water, pharmaceuticallyacceptable fats and oils, alcohols or other organic solvents, includingesters, emulsions, syrups or elixirs, suspensions, solutions and/ orsuspension reconstituted from non-effervescent preparationsreconstituted from effervescent granules. Such liquid dosage forms maycontain, for example, suitable solvents, preservatives, emulsifyingagents, suspending agents, diluents, sweeteners, thickeners and meltingagents. Parenteral and intravenous forms may also include isotonic saltsand other materials to make them compatible with the type of injectionor delivery system chosen.

[0111] For administration of an antibody to BMP-2 and/or BMP-4, thepharmaceutically acceptable carrier will usually be an aqueous solution,such as normal saline or phosphate-buffered saline (PBS), Ringer'ssolution, lactate-Ringer's solution, or any isotonic physiologicallyacceptable solution for administration by the chosen means. In additionto additives for adjusting pH or tonicity, the antibody may bestabilized against aggregation and polymerization with amino acids andnon-ionic detergents, polysorbate, and polyethylene glycol. Optionally,additional stabilizers may include various physiologically-acceptablecoarbohydrates and salts. Also, polyvinylpyrrolidone may be added inaddition to the amino acid. Suitable therapeutic immunoglobulinsolutions, which are stabilized for storage and administration to humansare described in U.S. Pat. No. 5,945,098. Other agents, such as humanserum albumin (HAS), may be added to the pharmaceutical composition tostabilize the antibody conjugates.

[0112] The method of administration can be any suitable method thateffectively alleviates the particular cancer being treated. Possiblemethods of administration are oral, rectal, parenteral, enterical,subcutaneous, transdermal, peritoneal, intratumoral, or intravenous.

[0113] Any suitable dosage of the compounds may be given in the methodof the invention. Dosage levels and requirements are well-recognized bythose of ordinary skill in the art. As one of ordinary skill in the artwill appreciate, an amount constituting an effective amount will varydepending on particular factors. For instance, specific dosage andtreatment regimens will depend on facts such as the patient's generalhealth profile, the type of cancer being treated, the severity andcourse of the patient's disorder, other therapeutics being administeredto treat the cancer, and the judgment of the treating physician.

[0114] The present invention also provides kits for treating cancerusing BMP-2 activity inhibitors. For example, such kits can comprise anyone or more of the following materials: packaging material, at least onetype of BMP-2 activity inhibitor and/or at least one type of BMP-4activity inhibitor, and instructions regarding dosage, method ofadministration, or the like for using the inhibitor to treat cancer.

[0115] Detection of BMP-2 to Aid in Diagnosis of Cancer

[0116] In addition to its therapeutic aspects, the present inventionalso relates to a diagnostic method for detecting the presence ofelevated levels of BMP-2 and/or BMP-4 in the patient. Applicant hasshown that BMP-2 is expressed in many common cancers. In addition, geneexpression of BMP-4, a protein that is highly homologous to BMP-2 andhas the same biological activity as BMP-2, has been detected in lungcancer tumors. BMP-4 shares 92% homology with BMP-2. Elevated levels ofBMP-2 and/or BMP-4 can be detected in various biological samples inmammals, preferably humans. Applicants have shown the presence of BMP-2in the blood serum of a human patient with cancer. Biological samples,including but not limited to blood, vitreous humor, sputum, aqueoushumor, synovial fluid, urine, ascites, and tissue, will be drawn fromthe patient using standard techniques. Particularly preferred are serumsamples.

[0117] The measurement of BMP-2 and/or BMP-4 levels may be monitoredusing any method possible to detect BMP-2 and/or BMP-4 in biologicalsamples. Immunoassays, such as Enzyme Linked Imnmunological Assay(ELISA), Western blots, immunoprecipitation, in situimunohistochemistry, and immunofluorescence assays are preferred. ELISAis particularly preferred. For a review of general immunoassays, seeStites, D. P., et al., eds., Basic and Clinical Immunology, 8^(th) ed.(Appleton & Lange, Norwalk, Conn.) (1994). Immunological binding assays(or immunoassays) typically use an antibody that specifically binds to aprotein or proteins of choice, BMP-2 and/or BMP-4, in this case. Theantibody is generally fixed to a substrate such as a plate or a columnvia covalent or non-covalent linkages (e.g., streptavidin, protein A,protein G, secondary antibodies). Immunoassays also often use a labelingagent to specifically bind to and label the complex formed by theantibody and antigen. The labeling agent may be a labeled anti-BMP-2antibody and/or a labeled anti-BMP-4 or a labeled antibody thatrecognizes both BMP-2 and BMP-4. Alternatively, the labeling agent maybe a third moiety, such as a secondary antibody, that specifically bindsto the antibody/antigen complex.

[0118] The immunoassays of this invention may be competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In a “sandwich” assay, forexample, the anti-BMP-2 antibodies can be bound directly to a solidsubstrate on which they are immobilized. These immobilized antibodiesthen capture BMP-2 in the test sample. BMP-2 thus immobilized is thenbound by a labeling agent, such as a second antibody bearing a label.The assay formats may also be performed with anti-BMP-4 antibodies orwith antibodies that recognize both BMP-2 and BMP-4. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety. Methods of binding molecules to a solid support,either covalently or non-covalently, are well known to those of skill inthe art. A variety of solid supports known to those of skill in the art,e.g., plate, columns, dipsticks, membranes, and the like, can be usedwith the present invention.

[0119] In competitive assays, the amount of BMP-2 and/or BMP-4 ismeasured indirectly by measuring the amount of a known modified BMP-2and/or BMP-4 displaced from a BMP-2 or BMP-4 antibody by the unknownBMP-2 and/or BMP-4 in a sample. In one example of a competitive assay, aknown amount of modified BMP-2 is added to a sample and the sample isthen contacted with an anti-BMP-2 antibody. The amount of known modifiedBMP-2 bound to the antibody is inversely proportional to theconcentration of BMP-2 in the sample. The amount of modified BMP-2 maybe detected by providing a labeled modified BMP-2 molecule.

[0120] The label used in the assay is not a critical aspect of theinvention, so long as it does not significantly interfere with thespecific binding antibody used in the assay. The detectable group can beany material having a detectable physical or chemical property. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical, or chemical means.Examples of such labels are magnetic beads, fluorescent dyes,radiolabels, enzymes, and calorimetric labels such as colloidal gold orcolored glass or plastic beads.

[0121] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions. Non-radioactive labels are often attached byindirect means. Generally, a ligand molecule, such as biotin, iscovalently bound to the molecule. The ligand then binds to anothermolecule, such as streptavidin, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize BMP-2 and/or BMP-4. The molecules can also be conjugateddirectly to a signal generating compound, e.g., by conjugation with anenzyme or fluorophore.

[0122] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers or the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally, simple colorimetriclabels may be detected simply by observing the color associated with thelabel.

[0123] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies.

[0124] Results

[0125] Experimental results supporting the above uses of BMP-2 and/orBMP-4 and their inhibitors are set forth in detail below. All of theexperimental methods mentioned in this section, such as representationaldifference analysis, Western blot assays, and immunohistochemicalstudies, are described in detail in the Examples section that follows.

[0126] Identification of BMP-2 Using RDA Subtraction Technique:

[0127] Initially, Applicant performed representational differenceanalysis (RDA) on cDNA derived from normal and cancerous lung tissuesamples to identify genes that were uniquely or highly expressed inhuman lung cancer in comparison to normal tissue. RDA has been describedin the literature and allows detection of differences in gene expressionbetween two similar populations. It involves exposing digested testercDNA ligated to a primer to high concentrations of similarly digestedbut non-primer bearing driver cDNA, melting the tester and driver cDNA,and allowing them to hybridize. Subsequent PCR results in exponentialamplification of the target cDNA of the tester that hybridizes to othertester cDNA. (Hubank, M., Nucleic Acids Research 22:5640-5648 (1994))Here, Applicant used a non-small cell lung carcinoma (NSCLC) as thetester and immortalized human bronchial epithelial (IHBE) cells as thedriver. IHBE cells rather than normal lung tissue were used, as IHBEcells proliferate at a rate that is more similar to human lungcarcinomas than to normal lung tissue. Thus, Applicant avoidedidentifying genes involved in the proliferation cascade but that werenot by themselves transforming.

[0128] After two rounds of subtraction, several distinct bands, whichwere cloned and sequenced, were present in the amplified tester cDNA.(FIG. 1b) A BLAST data base search identified BMP-2 expression in thelung tumor tissue specimen as well as expression of alpha-1-antitrypsin,cytokeratin 6, and lambda light. (FIG. 1c)

[0129] Expression of BMP-2 In Various Cancer Tissue Specimens, CancerCell Lines, and Blood Serum from a Cancer Patient

[0130] Using reverse transcriptase polymerase chain reaction (RT-PCR),Western blots, and immunohistochemical assays to study the expression ofBMP-2, BMP-4 and their receptors in various tissue specimens and in celllines, Applicant found that BMP-2 was highly expressed in many types ofcancers. Applicant also detected gene expression of BMP-4 in human lungcancer tumor samples.

[0131] Applicant performed his initial experiments on normal andcancerous lung tissue and lung cancer cell lines. Using Western blotanalysis, Applicant found that the mature active 14 kD BMP-2 protein wasaberrantly expressed in almost all of the 25 non-small cell lungcarcinoma (NSCLC) tissue specimens examined There was little to noexpression of BMP-2 in 11 normal lung tissue specimens. A representativeWestern blot is shown in FIG. 3. An anti-actin immunoblot showed nearequal loading of the samples. (FIG. 3(b)) In addition, BMP-2 was foundto be highly expressed in all epithelial derived lung carcinomas ofwhich NSCLC is derived and in the rare malignant neuroendocrine tumor.(FIG. 3(c) and FIG. 3(e), Lane 4, respectively) Western blot analysis ofeach of the different cell types comprising NSCLC—adeno, squamous, largecell, and bronchoalveolar carcinomas—revealed that the level of BMP-2expression was not dependent on the cell type or whether the tumor waswell or poorly differentiated. In comparison, the level of BMP-2expression in benign lung tumors (FIG. 3(e), Lane 1) and inflammatorydiseases of the lung (FIG. 7(a), Lane 1) was very low, similar to thatseen in normal lung tissue, showing that BMP-2 is not an acute phaseprotein and that high levels of BMP-2 expression are indicative ofmalignant tumors. Neither BMP-4 nor BMP-7 expression was detected in thelung tissue specimens or the A549, H7249, IHBE, and NBE cell lines byWestern analysis. (FIG. 3(f)) But BMP-4 gene expression was detected inRT-PCR experiments on cancerous human lung tissue specimens. And BMP-4expression was detected by Western blot analysis in the tumors thatdeveloped in nude mice injected with A549 cells transfected withexpression vectors encoding either BMP inhibitors or BMP receptorinhibitors. The results of both of these experiments will be discussedin more detail below.

[0132] Applicant also tested for expression of BMP-2 in various lungcancer and normal cell lines. Although the mature BMP-2 protein wasdetected in the cell lysate of the A549 and H7249 human lung cancer celllines, the level of expression was not significantly different from thelevel of expression in the cell lysate of immortalized normal humanbronchial epithelial cells (IHBE). (FIG. 4(a)) Because BMP-2 is asecreted protein, Applicant also examined its expression in the cellculture media. A Western blot of the cell culture media showed the A549and H7249 cell lines secreted a 43 kD BMP-2 precursor protein. (FIG.4(b), Lanes 2-3) This BMP-2 precursor was not detected in the media fromeither the IHBE or normal bronchial epithelial (NBE) cells (FIG. 4(b),Lanes 4-5).

[0133] Immunohistochemistry studies of patient derived NSCLC alsolocalized the expression of BMP-2 to the cancer cells (FIG. 5(a)).Absorbing the anti-BMP-2 antibody with recombinant human BMP-2completely inhibited staining of the tumors (FIG. 5(b)). BMP-2expression was not detected in normal lung tissue byimmunohistochemistry.

[0134] Applicant turned next to receptors and found that normal andcancer lung tissue specimens and cell lines express both type IA and IBBMP receptors. The lung cancer and normal lung tissue specimens expressa 55 kD and 44 kD type IA BMP-2 receptor. The tumor specimens expressedpredominately the 55 kD receptor, while normal lung tissue specimensexpressed a higher percentage of the 44 kD receptor. The A549, H7249,and IHBE cells only expressed a 44 kD type IA BMP receptor. (FIG. 4(c))The tissue specimens and cell lines expressed a 44 kD type IB BMPreceptor with normal lung tissue demonstrating more expression than thatof the tumor specimens. (FIG. 4(d))

[0135] Similar to their findings with lung tissue, Applicants found thatBMP-2 was expressed in many other common human malignancies but not intheir corresponding normal tissues. Western blot analysis revealed thatBMP-2 was overexpressed in breast, bladder, colon, endometrial, omental,and kidney carcinomas with low levels of BMP-2 expression in thecorresponding normal tissue. (FIGS. 6(a) and (b)). BMP-2 was also foundto be expressed in ovarian (FIG. 6(b), lane 3), mesothelioma (FIG. 3(e),lane 2), thyroid, hepatoma, and testicular carcinoma.

[0136] BMP-2 and its receptors were also examined in both primary andmetastatic carcinomas that were surgically removed from patients. BMP-2was found to be highly expressed in kidney tumors that had metastasizedto the lung, a metastatic breast cancer to chest wall cavity, and aNSCLC lung tumor that had metastasized to a regional lymph node. (FIG.7(a)) The BMP IA receptor was expressed equally between the primary andmetastatic carcinomas and the corresponding normal tissue (FIG. 7). TheBMP IB receptor was expressed in all metastatic and primary tumorsexamined. (FIG. 7) The BMP IB receptor, in contrast to the BMP IAreceptor, was not expressed in all the corresponding normal tissues.While it was expressed in normal lung tissue with slight expression innormal endometrium it was not expressed in normal kidney, colon, andomentum. (FIG. 7(f)) Interestingly, the IB receptor was expressed inboth primary and metastatic renal carcinoma, but not in normal kidneytissue. (FIG. 7(f), Lane 6)

[0137] BMP-2 expression was also found in blood serum samples from lungcancer patients. (FIG. 8)

[0138] Processing of Inactive BMP-2 Precursors

[0139] Because BMP precursors are proteolytically activated byproprotein convertases, Applicant studied whether BMP-2 could beprocessed following secretion, hypothesizing that secreted BMP-2precursors from tumor cells may be processed by cells present in thetumor stroma. Because leukocytes normally infiltrate lung and furinconvertase is ubiquitously expressed, the ability of leukocytes tocleave proprotein BMP-2 secreted from A549 cells was examined. First,Applicant determined that the furin convertase is expressed in humanleukocytes isolated from whole blood. (FIG. 9(c)). Human leukocytes wereincubated with A549 cell culture media containing the BMP-2 precursorprotein. A Western blot of the incubated media samples was probed withan anti-human BMP-2 precursor antibody that recognizes its C-terminalend. The 45 kD BMP-2 precursor protein was consistently decreasedfollowing incubation with the leukocytes (FIG. 9(a)). By probingimmunoblots with an anti-human BMP-2 antibody that recognizes itsN-terminal end, Applicant identified a 31 kD BMP-2 product present onlyin the media samples incubated with leukocytes. (FIG. 9(b)) This datashows that BMP-2 precursor proteins are cleaved by human leukocytes.

[0140] Effect of BMP-2 on Tumors and Cancer Cell Lines

[0141] After determining that BMP-2 was highly expressed in most commoncancers, Applicant performed experiments to show that BMP-2 causescancer invasion and metastasis. Applicant performed experiments withlung cancer cell lines and with nude mice injected with A549 cells.

[0142] The experiments with the nude mice showed that BMP-2 treatmentenhances blood vessel formation around tumors from nude mice injectedwith A549 cells. Some of the mice were co-injected with BMP-2. Grossobservations of tissue harvested after six days showed that the additionof recombinant BMP-2 to developing tumors in nude mice caused increasedblood vessel formation. (FIG. 10) Tissue was also stained with anti-CD31 antibody which recognizes endothelial cells. A person blind to howthe tumors were created then observed the tissue through a microscopeand counted the number of vessels that had formed in the tumor. Thisdata showed that BMP-2 caused a statistically significant increase inthe number of blood vessels in the tumor. (FIG. 11)

[0143] Other studies showed that addition of BMP-2 to cancer cell linesincreased expression of vascular endothelial growth factor (VEGF) andthe oncogene Sonic Hedgehog. VEGF is the most potent angiogenic factorand is though to be essential for tumor growth and metastasis. (Folkman,J. J. Nat'l Cancer Inst. 82:4 (1990); Zetter, B. Annual Rev. Med. 49:407(1998); Ferrara, N. Current Topics Microbiol. Immunol. 237:1 (1999))Transgenic mice studies have confirmed that overexpression of sonichedgehog can cause tissue-targeted cancer. (Oro, A. E., et al., “Basalcarcinomas in mice overexpressing sonic hedgehog” Science 276: 817-21(1997)) The addition of recombinant BMP-2 to human aortic endothelialcells in culture caused an increase in VEGF secretion as determined byELISA performed on the cell culture media. The concentration of VEGF inthe cell culture media before treatment with BMP-2 was 11.2 pg/ml. TheVEGF concentration after treatment with 0.500 pg/ml BMP-2 was 233.0pg/ml and after treatment with 1 ng/ml BMP-2 was 2,969.0 pg/ml. Theaddition of increasing amounts of BMP-2 to lung A549 lung cancer cellsgrowing in culture also caused a dose responsive increase in theexpression of the oncogene Sonic Hedgehog. (FIG. 12)

[0144] In addition, Applicants showed that BMP-2 stimulates themigration and invasion of the human lung cancer cell lines A549 andH7249. In one assay, recombinant BMP-2 caused a dose responsive increasein migration of cells from transwell migration chambers. (FIG. 13(a)) Inanother, BMP-2 stimulated the migration of A549 and H7249 cells culturedon glass cover slips toward Affi-blue agarose beads containingrecombinant BMP-2. (FIG. 13(c) and (d)) In addition, using transwellchambers coated with Matrigel, Applicants also showed that recombinantBMP-2 caused a dose responsive increase in the invasion of both A549 andH7249 cells. (FIG. 13(e))

[0145] Effects of Inhibiting BMP-2 Expression

[0146] After finding that BMP-2 enhances cancer invasion and growth,Applicant conducted experiments to determine whether inhibitors of theactivity of BMP-2, including anti-BMP-2 antibodies, could be used totreat cancer. In these studies, recombinant mouse noggin (R & D Systems,Minneapolis, Minn.) was used as a representative inhibitor. Noggin, anatural inhibitor of BMP-2, is a secreted protein that binds BMP-2 andBMP-4, thereby preventing their activation of the BMP receptors.(Weaver, M., et al., Development 126: 4005-4115 (1999); Zimmerman, L.B., et al., Cell 86: 599-606 (1996); Tucker, A.S., et al., Science 282:1136-1138 (1998); Capdevilla, J., et al., Developmental Biology 197:205-217 (1998); Brunet, L. J., et al., Science 280: 1455-1447 (1998))Mouse and human noggin are 98% homologous.

[0147] The effects of BMP-2 and noggin on tumor growth in vivo wasexamined by co-injecting the A549 cells subcutaneously into nude micewith Affi-Blue agarose beads coated with either albumin, recombinanthuman BMP-2, or recombinant human noggin. The animals were thensacrificed and tumors measured at 12 or 19 days. Inhibiting BMP-2activity with noggin resulted in a statistically significant decrease intumor growth. Addition of BMP-2 resulted in a statistically significantincrease in tumor growth. (FIG. 14)

[0148] When added to A549 cells noggin decreased the expression of VEGFand sonic hedgehog (FIGS. 12 and 15). Noggin also decreasedproliferation of A549 cells growing in culture.

[0149] Applicant also found that noggin completely inhibited the abilityof BMP-2, discussed above, to enhance the migration of the A549 cells ina transwell chamber. (FIG. 13(b))

[0150] Applicant also studied the effects of an anti-BMP-2 antibody ontumor growth in vivo. This was examined by co-injecting the A549 cellssubcutaneously into nude mice with either the anti-BMP-2 antibody orwith an isotype control antibody. The animals were sacrificed and tumorsharvested after three weeks. The addition of the anti-BMP-2 antibodyresulted in a statistically significant decrease in tumor growth.

[0151] Gene Expression of BMP-4 in Human Lung Cancer Tumor Specimens

[0152] In addition to his findings regarding BMP-2, Applicant detectedBMP-4 expression in human lung cancer tumor samples. Sequencing of cDNAobtained from RT-PCR performed on human lung cancer tumors revealed theexpression of BMP-4 in nine out of ten samples examined. As is discussedabove, BMP-4 is highly homologous to BMP-2; it is inhibited by the sameinhibitors that antagonize BMP-2 and binds to and activates the samereceptors that BMP-2 activates.

[0153] Expression of BMP-4 in Nude Mice

[0154] Using Western blot analysis, Applicant also found expression ofBMP-4 in the tumors of nude mice that had been injected either with 1)A549 cells transfected with expression vectors containing noggin, whichinhibits both BMP-2 and BMP-4, 2) A549 cells transfected with expressionvectors containing BMP receptor antagonists or 3) A549 cells transfectedwith expression vectors containing green fluorescent protein (GFP).Applicant compared the signals on the Western blot corresponding to thetumors from mice injected with the transfected cells to the signal forthe control-recombinant BMP-4. There was a strong signal for the tumorsresulting from the cells transfected with the noggin expression vectorand a fair signal for the tumors resulting from the cells transfectedwith the GFP expression vector.

EXAMPLES Example 1

[0155] Identification of BMP-2 Using Representational DifferenceAnalysis (RDA) Subtraction Technique

[0156] Representational difference analysis (RDA) subtraction techniquewas used to identify genes highly expressed in a non-small cell lungcarcinoma obtained from a patient (tester) in comparison to normalbronchial human epithelial cells (driver). The technique for RDAdescribed in the following references was followed: Holmes, M. L., etal., Molecular and Cellular Biology 19: 4182-4190 (1999); Hubank, M.,Nucleic Acids Research 22:5640-5648 (1994). Normal human bronchialepithelial cells were purchased from Clonetics, BioWhitaker(Walkersville, Md.) and were maintained in serum free media. Humantissue specimens were obtained directly from the operating room andimmediately frozen in liquid nitrogen. Tissue was stored in liquidnitrogen at −70 C.

[0157] To perform RDA, MRNA was purified from the samples using Oligo dTcolumns (Pharnacia, Peapack, N.J.) according to the manufacturer'sinstructions and cDNA was then obtained using the Pharmacia Time SavercDNA synthesis kit also according to the manufacturer's instructions.cDNA was digested with Sau3A I endonuclease, R-linker ligated, andamplified by PCR. The R-linkers were removed and J-linkers ligated tothe tester. The driver and tester cDNA were hybridized at 67 C. for 20hours (driver in excess 100:1) and the subtracted tester cDNA amplifiedby PCR. A second round of subtraction was performed using N-linkers(driver in excess 800,000: 1). The amplified PCR products were clonedinto blue script and sequenced using a EBI Prism 377 DNA sequencer.Known genes corresponding to the subtracted tumor cDNA were identifiedby a BLAST database search.

Example 2

[0158] Detection of Over-Expression of BMP and BMP Receptors in VariousCancer Tissue Specimens and Lung Cancer Cell Lines

[0159] Applicant detected expression of BMP and BMP receptors in anumber of normal and cancerous tissue specimens and cells. As describedabove, all human tissue specimens were obtained directly from theoperating room and immediately frozen in liquid nitrogen and stored at−70° C. Normal human bronchial epithelial (NBE) cells were purchasedfrom Clonetics, BioWhitaker (Walkersville, Md.) and were maintained inserum free media. Immortalized human bronchial epithelial (IHBE),BEAS-2B, cells were derived from normal bronchial epithelial cellsimmortalized with an adenovirus-12-5V40 hybrid virus (32). A549 andH7249 are highly invasive human lung cancer cell lines. The cell lineswere cultured in 5% fetal bovine serum (FBS) in Dulbecco's ModifiedEagles medium (DME) containing penicillin, streptomycin, and glutaminewith 5% pCO2 at 37° C. Western blot analysis was used to detectexpression of the BMP ligand and its receptors in all of these samples.Immunohistochemistry studies were performed to detect BMP in non-smallcell lung carcinoma samples and normal lung tissue samples derived frompatients.

[0160] Western Blot Analysis

[0161] In preparation for Western blot analysis, cells were lysed in amodified RIPA buffer containing 150 ml NaCl, 50 ml tris, pH 7.5, 1% NP40, 10% deoxycholic acid, and protease inhibitor cocktail fromCalbiochem. Tissue specimens were sonicated on ice in the same modifiedRIPA buffer. The protein concentration of the resulting samples wasmeasured using the Bradford assay technique. Recombinant human BMP-2,purchased from R & D Systems and reconstituted in PBS with gelatin,served as a control. Total cellular protein of the samples andrecombinant human BMP-2 were analyzed by SDS-PAGE, transferred tonitrocellulose filter (Schleicher and Schuell, Keene, N.H.) at 35 V for16 hours at 4° C. and then incubated with the desired primary antibody.Specific proteins were detected using the enhanced chemiluminescencesystem (Amersham, Arlington Heights, Ill.).

[0162] The primary antibodies that were used included mouse anti-humanBMP-2, goat anti-human BMP-4, goat anti-human BMP-7, goat anti-humantype IA BMP receptor, and goat anti-human type IB BMP-2 receptor. All ofthese antibodies, except the goat anti-human BMP-7 were purchased from R& D Systems in Minneapolis, Minn. The goat anti-human BMP-7 antibodywere obtained from Santa Cruz (Santa Cruz, Calif.).

[0163] Immunohistochemistry Analysis

[0164] To perform immunohistochemistry analysis, four micronCryostat-cut sections were air dried before being fixed in cold acetonefor 10 minutes. Sections were washed in cold 0.5 M PBS and intrinsicperoxidase was quenched with 0.03% periodic acid for 20 minutes at roomtemperature. Sections were then rinsed in cold PBS and 0.5% BSA/PBS wasapplied to the slides for 15 minutes in a humid chamber. BiotinylatedBMP-2/4 (R & D Systems) was applied at a 1:25 dilution in 1% BSA/PBS andincubated overnight at 4 C. Two slides were run as negative controls.One slide was incubated with biotinylated BMP-2 preabsorbed withrecombinant human BMP-2 at 1:10 Molar ratio. As a second negativecontrol slide samples were incubated overnight at 4 C. with normalrabbit serum. Slides were washed with cold PBS and incubated for onehour in Streptavidin horseradish peroxidase (Dako) at a 1:500 dilutionin 1% BSA/PBS. Slides were then counterstained in 0.7% Toluidine Blue.

Example 3

[0165] Detection of Processing of Mature BMP-2 by Human Leukocytes

[0166] Cell culture media from the A549 cells was incubated withleukocytes isolated from whole blood for 16 hours. Then, a Western blotwas performed, as described above, on the cell culture media. Mouseanti-human BMP-2 antibody (#MAB355, R & D Systems, Minneapolis, Minn.)was the primary antibody used to detect the C-terminal end of BMP-2.Goat anti-human BMP-2 (Research Diagnostics, Flanders, N.J.) was used todetect the N-terminal end of BMP-2. A Western blot of the leukocytes wasalso performed with an anti-furin primary antibody to determine thathuman leukocytes express furin convertase.

Example 4

[0167] Analysis of the Effect of BMP-2 and Noggin on Tumor Growth andTumor Vasculature In Vivo

[0168] Nude mice studies were conducted to determine the effect of BMP-2and one of its inhibitors, noggin, on tumor growth and tumorvasculature. 10⁶ A549 cells were injected subcutaneously into nude micewith Affi-Blue agarose beads coated with albumin, recombinant humanBMP-2 or recombinant mouse noggin. Both of these recombinant proteinswere purchased from R & D Systems and were reconstituted in PBS withgelatin. Coating of Affi-Blue agarose beads with BMP-2 and noggin hasbeen described in the literature. (Abe, E., et al., J. Bone Miner Res.15: 663-673 (2000); Tucker, A. S., et al., Science 282: 1136-1138(1998); Zimmerman, L. B., et al., Cell 86: 599-606 (1996)) In brief, 25ug of Affi-blue agarose beads were incubated with 100 ug/ml albumin,recombinant human BMP-2, or recombinant noggin for 2 hours and thenwashed 3 times with PBS immediately prior to use. In separateexperiments the beads were not washed prior to injection. The coatedbeads were injected with the A549 cells into nude mice subcutaneously.To assess tumor growth after 12 or 19 days the length, width, and depthof the tumors were measured in mm. To assess tumor vasculature, tissueincluding a tumor was harvested after six days. Gross observations ofthe tissue were made. In addition, the tissue was stained with anti-CD31 antibody, which recognizes endothelial cells. Vessels in five highpower fields were counted by a person blinded to how the tumors werecreated.

[0169] Nude mice studies were also conducted to determine the effect ofan anti-BMP-2 antibody on tumor growth. As with the experiments withnoggin and BMP-2 described above, 10⁶ A549 cells were co-injectedsubcutaneously into nude mice with either the anti-BMP-2 antibody(Genetics Institute, Andover, Mass.) or with an isotype controlantibody. Tumors were harvested after three weeks and tumor growthassessed by measuring the length, width, and depth of the tumors in mm.

Example 5

[0170] Effect of BMP-2 and Noggin on VEGF and Sonic Hedgehog Expression

[0171] Western blot analvsis of VEGF and sonic hedgehog in presence ofBMP-2 and noggin

[0172] Western blots, as described above, were performed on totalcellular protein samples and cell culture media samples. The primaryantibodies used to detect VEGF and sonic hedgehog were anti human VEGFfrom R & D Systems (Minneapolis, Minn.) and anti human sonic hedgehogfrom Santa Cruz (Santa Cruz, Calif.), respectively.

[0173] ELISA of VEGF in presence of BMP-2 and Various Concentrations ofNoggin

[0174] The sandwich ELISA method was used to determine VEGFconcentrations in the cell culture media of A549 cells treated withnoggin and in the cell culture media of human aortic endothelial cellstreated with BMP-2. 100 ul of the monoclonal capture antibody, dilutedin carbonate buffer (sodium bicarbonate, sodium carbonate, pH 9.0), wasadded to each well of a MaxiSorb Nunc-immuno plate and incubatedovernight at 4 C. The plates were washed two times with washing buffer(1× PBS with 0.0005% tween-20). Then, 200 ul of blocking buffer (1× PBSwith 1% BSA) was added per well and incubated for 2 hours at roomtemperature. The plates were then washed 4 times with washing buffer.

[0175] The recombinant protein standards and samples (100 ul per well)were added and the plate was then incubated overnight at 4 C. The plateswere washed 5 times with washing buffer. The biotinylated detectionantibody was diluted in incubation buffer (1× PBS with 10% fetal bovineserum) for a final concentration of 1 ug/ml. 100 ul of the detectionantibody was added per well and incubated for 1 hour on a shaker at roomtemperature. The plates were washed 6 times with washing buffer and 100ul of streptavidin-HRP conjugate (1:10,000) was added per well. Theplates were incubated for 45 minutes at room temperature on a shaker andthen washed 6 times with washing buffer. 100 ul/well of the substratereagent (0.2 M citrate buffer, 1 mg/ml o-phenylenediaminedihydrocholoride (OPG), 3% hydrogen peroxide) was added and covered withaluminum foil for ten minutes. The reaction was stopped with 100 ul/wellof 2M sulfuiric acid and absorbance determined using an automated platereader with a 490/690 filter. The protein concentration was thendetermined from the standard curve.

Example 6

[0176] Analysis of Effect of Noggin on A549 Cell Growth

[0177] A549 cell cultures grown in DME media with fetal calf serum weretreated with recombinant mouse noggin. (R & D Systems) Using ahemacytometer, cell counts were then taken after two days and after fourdays. Growth suppression was seen at 1 ng/ml noggin.

[0178] Example 7

[0179] Identification of BMP-2 as a Stimulant of Human Lung Cancer CellMigration and Invasion

[0180] Migration Assay In Monolayer Culture

[0181] To detect BMP-induced migration in a monolayer culture,recombinant human BMP-2 (R & D systems, Minneapolis, Minn.) was coatedto Affi-Blue agarose beads (Bio Rad, Hercules, Calif.) as described inthe literature. (Vainio, S.; et al., Cell 75: 45-58 (1993); Sloan, A.J., et al., Arch Oral Biol. 44: 149-156 (1999)) Briefly, 100 ml of theAffi-Blue agarose beads were incubated with either 10 ml of recombinantBMP2 reconstituted in PBS with gelatin (100 mg/ml) or PBS alone at 37°C. for 2 hours, washed with PBS, and reconstituted with 40 ml of PBS.Glass cover slips were coated with serum free media containing BSA,fibronectin and collagen (32) and 50,000 cells were plated per coverslip in serum free media. Two microliters of the Affi-Blue agarose beadscoated with recombinant BMP-2 or dilution buffer were placed in linearfashion next to the cover slips.

[0182] Chemotactic Assay

[0183] In the chemotactic assay, fifty thousand cells were placed in theupper chamber of an 8 micron transwell migration chamber (BectonDickinson, Bedford, Mass.) and 300 ml of serum free media with 0 ng/ml,1 ng/ml, 10 ng/ml, 100 ng/ml, 500 ng/ml, or 1000 ng/ml recombinant humanBMP-2 placed in the lower well. After 24 hours the filters were thenremoved and the top of the filter wiped with a cotton swab and the cellsthat migrated through the filters were stained with Syto-16intercalating dye. Five high power fields were counted using fluorescentmicroscopy. To show that noggin inhibits BMP-2 induced migration, theexperiment was also performed with each of the following in the lowerwell of the transwell chamber: media alone, recombinant BMP-2 (500ng/ml), and noggin (10 ug/ml) with recombinant BMP-2 (500 ng/ml).

[0184] Matrigel Invasion Assay

[0185] Invasion was studied using transwell chambers coated with 100 mlof Matrigel (Becton Dickinson). Fifty thousand cells were placed in theupper chamber and 300 ml of serum free media with 0 ng/ml, 10 ng/ml, 100ng/ml, 500 ng/ml, or 1000 ng/ml recombinant BMP-2 placed in the lowerwells. After 48 hours the Matrigel was removed and cells that hadmigrated through the filter were stained with Syto-16 intercalating dyeand 5 high power fields counted using fluorescent microscopy.

Example 8

[0186] Detection of Gene Expression of BMP-4 in Human Lung CancerSpecimens Using RT-PCR and Sequencing

[0187] Reverse transcriptase polymerase chain reaction was performedusing standard techniques well known in the art. The forward primer wasacgagagctctcactggtcc (SEQ ID No: 15). The reverse primer wascattccggattacatgaggg (SEQ ID No: 16). The chain reaction consisted ofdenaturation at 95 C. for 1 min, annealing at 54 C. for 1 min, andextension at 72 C. for 2 min with 33 cycles. The resulting cDNA wassequenced at a core facility at the University of Medicine and Dentistryof New Jersey, using an automated sequencer.

Example 9

[0188] Detection of Expression of BMP-4 in Tumors of Nude Mice Injectedwith A549 Cells Transfected with Various Constructs

[0189] A549 cells were transfected with expression vectors that expressgreen fluorescent protein (GFP), bone morphogenetic protein receptor IAantagonist, bone morphogenetic protein receptor IB antagonist, andnoggin. 10⁶ of each of the transfected cells were then injectedsubcutaneously into nude mice. The resulting tumors were harvested afterthree weeks. Western blots, as described above, were performed on totalcellular protein samples. The primary antibody used was goat anti-humanBMP-4 and was purchased from R & D Systems. The recombinant BMP-4 usedas a control was a human recombinant and was also purchased from R & DSystems.

1 18 1 1547 DNA Homo sapiens source (1)..(1547) Homo sapiens Taxon9606 1ggggacttct tgaacttgca gggagaataa cttgcgcacc ccactttgcg ccggtgcctt 60tgccccagcg gagcctgctt cgccatctcc gagccccacc gcccctccac tcctcggcct 120tgcccgacac tgagacgctg ttcccagcgt gaaaagagag actgcgcggc cggcacccgg 180gagaaggagg aggcaaagaa aaggaacgga cattcggtcc ttgcgccagg tcctttgacc 240agagtttttc catgtggacg ctctttcaat ggacgtgtcc ccgcgtgctt cttagacgga 300ctgcggtctc ctaaaggtcg acc atg gtg gcc ggg acc cgc tgt ctt cta gcg 353Met Val Ala Gly Thr Arg Cys Leu Leu Ala 1 5 10 ttg ctg ctt ccc cag gtcctc ctg ggc ggc gcg gct ggc ctc gtt ccg 401 Leu Leu Leu Pro Gln Val LeuLeu Gly Gly Ala Ala Gly Leu Val Pro 15 20 25 gag ctg ggc cgc agg aag ttcgcg gcg gcg tcg tcg ggc cgc ccc tca 449 Glu Leu Gly Arg Arg Lys Phe AlaAla Ala Ser Ser Gly Arg Pro Ser 30 35 40 tcc cag ccc tct gac gag gtc ctgagc gag ttc gag ttg cgg ctg ctc 497 Ser Gln Pro Ser Asp Glu Val Leu SerGlu Phe Glu Leu Arg Leu Leu 45 50 55 agc atg ttc ggc ctg aaa cag aga cccacc ccc agc agg gac gcc gtg 545 Ser Met Phe Gly Leu Lys Gln Arg Pro ThrPro Ser Arg Asp Ala Val 60 65 70 gtg ccc ccc tac atg cta gac ctg tat cgcagg cac tca ggt cag ccg 593 Val Pro Pro Tyr Met Leu Asp Leu Tyr Arg ArgHis Ser Gly Gln Pro 75 80 85 90 ggc tca ccc gcc cca gac cac cgg ttg gagagg gca gcc agc cga gcc 641 Gly Ser Pro Ala Pro Asp His Arg Leu Glu ArgAla Ala Ser Arg Ala 95 100 105 aac act gtg cgc agc ttc cac cat gaa gaatct ttg gaa gaa cta cca 689 Asn Thr Val Arg Ser Phe His His Glu Glu SerLeu Glu Glu Leu Pro 110 115 120 gaa acg agt ggg aaa aca acc cgg aga ttcttc ttt aat tta agt tct 737 Glu Thr Ser Gly Lys Thr Thr Arg Arg Phe PhePhe Asn Leu Ser Ser 125 130 135 atc ccc acg gag gag ttt atc acc tca gcagag ctt cag gtt ttc cga 785 Ile Pro Thr Glu Glu Phe Ile Thr Ser Ala GluLeu Gln Val Phe Arg 140 145 150 gaa cag atg caa gat gct tta gga aac aatagc agt ttc cat cac cga 833 Glu Gln Met Gln Asp Ala Leu Gly Asn Asn SerSer Phe His His Arg 155 160 165 170 att aat att tat gaa atc ata aaa cctgca aca gcc aac tcg aaa ttc 881 Ile Asn Ile Tyr Glu Ile Ile Lys Pro AlaThr Ala Asn Ser Lys Phe 175 180 185 ccc gtg acc aga ctt ttg gac acc aggttg gtg aat cag aat gca agc 929 Pro Val Thr Arg Leu Leu Asp Thr Arg LeuVal Asn Gln Asn Ala Ser 190 195 200 agg tgg gaa agt ttt gat gtc acc cccgct gtg atg cgg tgg act gca 977 Arg Trp Glu Ser Phe Asp Val Thr Pro AlaVal Met Arg Trp Thr Ala 205 210 215 cag gga cac gcc aac cat gga ttc gtggtg gaa gtg gcc cac ttg gag 1025 Gln Gly His Ala Asn His Gly Phe Val ValGlu Val Ala His Leu Glu 220 225 230 gag aaa caa ggt gtc tcc aag aga catgtt agg ata agc agg tct ttg 1073 Glu Lys Gln Gly Val Ser Lys Arg His ValArg Ile Ser Arg Ser Leu 235 240 245 250 cac caa gat gaa cac agc tgg tcacag ata agg cca ttg cta gta act 1121 His Gln Asp Glu His Ser Trp Ser GlnIle Arg Pro Leu Leu Val Thr 255 260 265 ttt ggc cat gat gga aaa ggg catcct ctc cac aaa aga gaa aaa cgt 1169 Phe Gly His Asp Gly Lys Gly His ProLeu His Lys Arg Glu Lys Arg 270 275 280 caa gcc aaa cac aaa cag cgg aaacgc ctt aag tcc agc tgt aag aga 1217 Gln Ala Lys His Lys Gln Arg Lys ArgLeu Lys Ser Ser Cys Lys Arg 285 290 295 cac cct ttg tac gtg gac ttc agtgac gtg ggg tgg aat gac tgg att 1265 His Pro Leu Tyr Val Asp Phe Ser AspVal Gly Trp Asn Asp Trp Ile 300 305 310 gtg gct ccc ccg ggg tat cac gccttt tac tgc cac gga gaa tgc cct 1313 Val Ala Pro Pro Gly Tyr His Ala PheTyr Cys His Gly Glu Cys Pro 315 320 325 330 ttt cct ctg gct gat cat ctgaac tcc act aat cat gcc att gtt cag 1361 Phe Pro Leu Ala Asp His Leu AsnSer Thr Asn His Ala Ile Val Gln 335 340 345 acg ttg gtc aac tct gtt aactct aag att cct aag gca tgc tgt gtc 1409 Thr Leu Val Asn Ser Val Asn SerLys Ile Pro Lys Ala Cys Cys Val 350 355 360 ccg aca gaa ctc agt gct atctcg atg ctg tac ctt gac gag aat gaa 1457 Pro Thr Glu Leu Ser Ala Ile SerMet Leu Tyr Leu Asp Glu Asn Glu 365 370 375 aag gtt gta tta aag aac tatcag gac atg gtt gtg gag ggt tgt ggg 1505 Lys Val Val Leu Lys Asn Tyr GlnAsp Met Val Val Glu Gly Cys Gly 380 385 390 tgt cgc tag tacagcaaaattaaatacat aaatatatat ata 1547 Cys Arg 395 2 396 PRT Homo sapiensmisc_feature (429)..(1127) Region TGF-beta propeptide 2 Met Val Ala GlyThr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val 1 5 10 15 Leu Leu GlyGly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys 20 25 30 Phe Ala AlaAla Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu 35 40 45 Val Leu SerGlu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys 50 55 60 Gln Arg ProThr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu 65 70 75 80 Asp LeuTyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp 85 90 95 His ArgLeu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe 100 105 110 HisHis Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr 115 120 125Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe 130 135140 Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala 145150 155 160 Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr GluIle 165 170 175 Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr ArgLeu Leu 180 185 190 Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp GluSer Phe Asp 195 200 205 Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln GlyHis Ala Asn His 210 215 220 Gly Phe Val Val Glu Val Ala His Leu Glu GluLys Gln Gly Val Ser 225 230 235 240 Lys Arg His Val Arg Ile Ser Arg SerLeu His Gln Asp Glu His Ser 245 250 255 Trp Ser Gln Ile Arg Pro Leu LeuVal Thr Phe Gly His Asp Gly Lys 260 265 270 Gly His Pro Leu His Lys ArgGlu Lys Arg Gln Ala Lys His Lys Gln 275 280 285 Arg Lys Arg Leu Lys SerSer Cys Lys Arg His Pro Leu Tyr Val Asp 290 295 300 Phe Ser Asp Val GlyTrp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr 305 310 315 320 His Ala PheTyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His 325 330 335 Leu AsnSer Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val 340 345 350 AsnSer Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala 355 360 365Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn 370 375380 Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 385 390 395 3 699DNA Homo sapiens source (1)..(699) Homo sapiens Taxon9606 3 atg gag cgctgc ccc agc cta ggg gtc acc ctc tac gcc ctg gtg gtg 48 Met Glu Arg CysPro Ser Leu Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 gtc ctg gggctg cgg gcg aca ccg gcc ggc ggc cag cac tat ctc cac 96 Val Leu Gly LeuArg Ala Thr Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 atc cgc ccg gcaccc agc gac aac ctg ccc ctg gtg gac ctc atc gaa 144 Ile Arg Pro Ala ProSer Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 cac cca gac cct atcttt gac ccc aag gaa aag gat ctg aac gag acg 192 His Pro Asp Pro Ile PheAsp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55 60 ctg ctg cgc tcg ctg ctcggg ggc cac tac gac cca ggc ttc atg gcc 240 Leu Leu Arg Ser Leu Leu GlyGly His Tyr Asp Pro Gly Phe Met Ala 65 70 75 80 acc tcg ccc ccc gag gaccgg ccc ggc ggg ggc ggg ggt gca gct ggg 288 Thr Ser Pro Pro Glu Asp ArgPro Gly Gly Gly Gly Gly Ala Ala Gly 85 90 95 ggc gcg gag gac ctg gcg gagctg gac cag ctg ctg cgg cag cgg ccg 336 Gly Ala Glu Asp Leu Ala Glu LeuAsp Gln Leu Leu Arg Gln Arg Pro 100 105 110 tcg ggg gcc atg ccg agc gagatc aaa ggg cta gag ttc tcc gag ggc 384 Ser Gly Ala Met Pro Ser Glu IleLys Gly Leu Glu Phe Ser Glu Gly 115 120 125 ttg gcc cag ggc aag aag cagcgc cta agc aag aag ctg cgg agg aag 432 Leu Ala Gln Gly Lys Lys Gln ArgLeu Ser Lys Lys Leu Arg Arg Lys 130 135 140 tta cag atg tgg ctg tgg tcgcag aca ttc tgc ccc gtg ctg tac gcg 480 Leu Gln Met Trp Leu Trp Ser GlnThr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 tgg aac gac ctg ggc agccgc ttt tgg ccg cgc tac gtg aag gtg ggc 528 Trp Asn Asp Leu Gly Ser ArgPhe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 agc tgc ttc agt aag cgctcg tgc tcc gtg ccc gag ggc atg gtg tgc 576 Ser Cys Phe Ser Lys Arg SerCys Ser Val Pro Glu Gly Met Val Cys 180 185 190 aag ccg tcc aag tcc gtgcac ctc acg gtg ctg cgg tgg cgc tgt cag 624 Lys Pro Ser Lys Ser Val HisLeu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 cgg cgc ggg ggc cag cgctgc ggc tgg att ccc atc cag tac ccc atc 672 Arg Arg Gly Gly Gln Arg CysGly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 att tcc gag tgc aag tgctcg tgc tag 699 Ile Ser Glu Cys Lys Cys Ser Cys 225 230 4 232 PRT Homosapiens 4 Met Glu Arg Cys Pro Ser Leu Gly Val Thr Leu Tyr Ala Leu ValVal 1 5 10 15 Val Leu Gly Leu Arg Ala Thr Pro Ala Gly Gly Gln His TyrLeu His 20 25 30 Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu Val Asp LeuIle Glu 35 40 45 His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu AsnGlu Thr 50 55 60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly PheMet Ala 65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly GlyAla Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu Leu ArgGln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys Gly Leu GluPhe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys Lys Gln Arg Leu Ser LysLys Leu Arg Arg Lys 130 135 140 Leu Gln Met Trp Leu Trp Ser Gln Thr PheCys Pro Val Leu Tyr Ala 145 150 155 160 Trp Asn Asp Leu Gly Ser Arg PheTrp Pro Arg Tyr Val Lys Val Gly 165 170 175 Ser Cys Phe Ser Lys Arg SerCys Ser Val Pro Glu Gly Met Val Cys 180 185 190 Lys Pro Ser Lys Ser ValHis Leu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 Arg Arg Gly Gly GlnArg Cys Gly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 Ile Ser Glu CysLys Cys Ser Cys 225 230 5 699 DNA Mus musculus gene (1)..(699) nog 5 atggag cgc tgc ccc agc ctg ggg gtc acc ctc tac gcc ctg gtg gtg 48 Met GluArg Cys Pro Ser Leu Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 gtcctg ggg ctg cgg gca gca cca gcc ggc ggc cag cac tat cta cac 96 Val LeuGly Leu Arg Ala Ala Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 atc cgccca gca ccc agc gac aac ctg ccc ttg gtg gac ctc atc gaa 144 Ile Arg ProAla Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 cat cca gaccct atc ttt gac cct aag gag aag gat ctg aac gag acg 192 His Pro Asp ProIle Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55 60 ctg ctg cgc tcgctg ctc ggg ggc cac tac gac ccg ggc ttt atg gcc 240 Leu Leu Arg Ser LeuLeu Gly Gly His Tyr Asp Pro Gly Phe Met Ala 65 70 75 80 act tcg ccc ccagag gac cga ccc gga ggg ggc ggg gga ccg gct gga 288 Thr Ser Pro Pro GluAsp Arg Pro Gly Gly Gly Gly Gly Pro Ala Gly 85 90 95 ggt gcc gag gac ctggcg gag ctg gac cag ctg ctg cgg cag cgg ccg 336 Gly Ala Glu Asp Leu AlaGlu Leu Asp Gln Leu Leu Arg Gln Arg Pro 100 105 110 tcg ggg gcc atg ccgagc gag atc aaa ggg ctg gag ttc tcc gag ggc 384 Ser Gly Ala Met Pro SerGlu Ile Lys Gly Leu Glu Phe Ser Glu Gly 115 120 125 ttg gcc caa ggc aagaaa cag cgc ctg agc aag aag ctg agg agg aag 432 Leu Ala Gln Gly Lys LysGln Arg Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 tta cag atg tgg ctgtgg tca cag acc ttc tgc ccg gtg ctg tac gcg 480 Leu Gln Met Trp Leu TrpSer Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 tgg aat gac ctaggc agc cgc ttt tgg cca cgc tac gtg aag gtg ggc 528 Trp Asn Asp Leu GlySer Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 agc tgc ttc agcaag cgc tcc tgc tct gtg ccc gag ggc atg gtg tgt 576 Ser Cys Phe Ser LysArg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180 185 190 aag cca tcc aagtct gtg cac ctc acg gtg ctg cgg tgg cgc tgt cag 624 Lys Pro Ser Lys SerVal His Leu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 cgg cgc ggg ggtcag cgc tgc ggc tgg att ccc atc cag tac ccc atc 672 Arg Arg Gly Gly GlnArg Cys Gly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 att tcc gag tgtaag tgt tcc tgc tag 699 Ile Ser Glu Cys Lys Cys Ser Cys 225 230 6 232PRT Mus musculus 6 Met Glu Arg Cys Pro Ser Leu Gly Val Thr Leu Tyr AlaLeu Val Val 1 5 10 15 Val Leu Gly Leu Arg Ala Ala Pro Ala Gly Gly GlnHis Tyr Leu His 20 25 30 Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu ValAsp Leu Ile Glu 35 40 45 His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys AspLeu Asn Glu Thr 50 55 60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp ProGly Phe Met Ala 65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly GlyGly Gly Pro Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln LeuLeu Arg Gln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys GlyLeu Glu Phe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys Lys Gln Arg LeuSer Lys Lys Leu Arg Arg Lys 130 135 140 Leu Gln Met Trp Leu Trp Ser GlnThr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 Trp Asn Asp Leu Gly SerArg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 Ser Cys Phe Ser LysArg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180 185 190 Lys Pro Ser LysSer Val His Leu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 Arg Arg GlyGly Gln Arg Cys Gly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 Ile SerGlu Cys Lys Cys Ser Cys 225 230 7 3547 DNA Homo sapiens source(1)..(3547) Taxon9606 7 cccgggtcag cgcccgcccg cccgcgctcc tcccggccgctcctcccgcc ccgcccggcc 60 cggcgccgac tctgcggccg cccgacgagc ccctcgcggcactgccccgg ccccggcccc 120 ggccccggcc ccctcccgcc gcaccgcccc cggcccggccctccgccctc cgcactcccg 180 cctccctccc tccgcccgct cccgcgccct cctccctccctcctccccag ctgtcccgtt 240 cgcgtc atg ccg agc ctc ccg gcc ccg ccg gcc ccgctg ctg ctc ctc 288 Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Leu Leu LeuLeu 1 5 10 ggg ctg ctg ctg ctc ggc tcc cgg ccg gcc cgc ggc gcc ggc cccgag 336 Gly Leu Leu Leu Leu Gly Ser Arg Pro Ala Arg Gly Ala Gly Pro Glu15 20 25 30 ccc ccc gtg ctg ccc atc cgt tct gag aag gag ccg ctg ccc gttcgg 384 Pro Pro Val Leu Pro Ile Arg Ser Glu Lys Glu Pro Leu Pro Val Arg35 40 45 gga gcg gca ggc tgc acc ttc ggc ggg aag gtc tat gcc ttg gac gag432 Gly Ala Ala Gly Cys Thr Phe Gly Gly Lys Val Tyr Ala Leu Asp Glu 5055 60 acg tgg cac ccg gac cta ggg gag cca ttc ggg gtg atg cgc tgc gtg480 Thr Trp His Pro Asp Leu Gly Glu Pro Phe Gly Val Met Arg Cys Val 6570 75 ctg tgc gcc tgc gag gcg cct cag tgg ggt cgc cgt acc agg ggc cct528 Leu Cys Ala Cys Glu Ala Pro Gln Trp Gly Arg Arg Thr Arg Gly Pro 8085 90 ggc agg gtc agc tgc aag aac atc aaa cca gag tgc cca acc ccg gcc576 Gly Arg Val Ser Cys Lys Asn Ile Lys Pro Glu Cys Pro Thr Pro Ala 95100 105 110 tgt ggg cag ccg cgc cag ctg ccg gga cac tgc tgc cag acc tgcccc 624 Cys Gly Gln Pro Arg Gln Leu Pro Gly His Cys Cys Gln Thr Cys Pro115 120 125 cag gag cgc agc agt tcg gag cgg cag ccg agc ggc ctg tcc ttcgag 672 Gln Glu Arg Ser Ser Ser Glu Arg Gln Pro Ser Gly Leu Ser Phe Glu130 135 140 tat ccg cgg gac ccg gag cat cgc agt tat agc gac cgc ggg gagcca 720 Tyr Pro Arg Asp Pro Glu His Arg Ser Tyr Ser Asp Arg Gly Glu Pro145 150 155 ggc gct gag gag cgg gcc cgt ggt gac ggc cac acg gac ttc gtggcg 768 Gly Ala Glu Glu Arg Ala Arg Gly Asp Gly His Thr Asp Phe Val Ala160 165 170 ctg ctg aca ggg ccg agg tcg cag gcg gtg gca cga gcc cga gtctcg 816 Leu Leu Thr Gly Pro Arg Ser Gln Ala Val Ala Arg Ala Arg Val Ser175 180 185 190 ctg ctg cgc tct agc ctc cgc ttc tct atc tcc tac agg cggctg gac 864 Leu Leu Arg Ser Ser Leu Arg Phe Ser Ile Ser Tyr Arg Arg LeuAsp 195 200 205 cgc cct acc agg atc cgc ttc tca gac tcc aat ggc agt gtcctg ttt 912 Arg Pro Thr Arg Ile Arg Phe Ser Asp Ser Asn Gly Ser Val LeuPhe 210 215 220 gag cac cct gca gcc ccc acc caa gat ggc ctg gtc tgt ggggtg tgg 960 Glu His Pro Ala Ala Pro Thr Gln Asp Gly Leu Val Cys Gly ValTrp 225 230 235 cgg gca gtg cct cgg ttg tct ctg cgg ctc ctt agg gca gaacag ctg 1008 Arg Ala Val Pro Arg Leu Ser Leu Arg Leu Leu Arg Ala Glu GlnLeu 240 245 250 cat gtg gca ctt gtg aca ctc act cac cct tca ggg gag gtctgg ggg 1056 His Val Ala Leu Val Thr Leu Thr His Pro Ser Gly Glu Val TrpGly 255 260 265 270 cct ctc atc cgg cac cgg gcc ctg gct gca gag acc ttcagt gcc atc 1104 Pro Leu Ile Arg His Arg Ala Leu Ala Ala Glu Thr Phe SerAla Ile 275 280 285 ctg act cta gaa ggc ccc cca cag cag ggc gta ggg ggcatc acc ctg 1152 Leu Thr Leu Glu Gly Pro Pro Gln Gln Gly Val Gly Gly IleThr Leu 290 295 300 ctc act ctc agt gac aca gag gac tcc ttg cat ttt ttgctg ctc ttc 1200 Leu Thr Leu Ser Asp Thr Glu Asp Ser Leu His Phe Leu LeuLeu Phe 305 310 315 cga ggg ctg ctg gaa ccc agg agt ggg gga cta acc caggtt ccc ttg 1248 Arg Gly Leu Leu Glu Pro Arg Ser Gly Gly Leu Thr Gln ValPro Leu 320 325 330 agg ctc cag att cta cac cag ggg cag cta ctg cga gaactt cag gcc 1296 Arg Leu Gln Ile Leu His Gln Gly Gln Leu Leu Arg Glu LeuGln Ala 335 340 345 350 aat gtc tca gcc cag gaa cca ggc ttt gct gag gtgctg ccc aac ctg 1344 Asn Val Ser Ala Gln Glu Pro Gly Phe Ala Glu Val LeuPro Asn Leu 355 360 365 aca gtc cag gag atg gac tgg ctg gtg ctg ggg gagctg cag atg gcc 1392 Thr Val Gln Glu Met Asp Trp Leu Val Leu Gly Glu LeuGln Met Ala 370 375 380 ctg gag tgg gca ggc agg cca ggg ctg cgc atc agtgga cac att gct 1440 Leu Glu Trp Ala Gly Arg Pro Gly Leu Arg Ile Ser GlyHis Ile Ala 385 390 395 gcc agg aag agc tgc gac gtc ctg caa agt gtc ctttgt ggg gct gat 1488 Ala Arg Lys Ser Cys Asp Val Leu Gln Ser Val Leu CysGly Ala Asp 400 405 410 gcc ctg atc cca gtc cag acg ggt gct gcc ggc tcagcc agc ctc acg 1536 Ala Leu Ile Pro Val Gln Thr Gly Ala Ala Gly Ser AlaSer Leu Thr 415 420 425 430 ctg cta gga aat ggc tcc ctg atc tat cag gtgcaa gtg gta ggg aca 1584 Leu Leu Gly Asn Gly Ser Leu Ile Tyr Gln Val GlnVal Val Gly Thr 435 440 445 agc agt gag gtg gtg gcc atg aca ctg gag accaag cct cag cgg agg 1632 Ser Ser Glu Val Val Ala Met Thr Leu Glu Thr LysPro Gln Arg Arg 450 455 460 gat cag cgc act gtc ctg tgc cac atg gct ggactc cag cca gga gga 1680 Asp Gln Arg Thr Val Leu Cys His Met Ala Gly LeuGln Pro Gly Gly 465 470 475 cac acg gcc gtg ggt atc tgc cct ggg ctg ggtgcc cga ggg gct cat 1728 His Thr Ala Val Gly Ile Cys Pro Gly Leu Gly AlaArg Gly Ala His 480 485 490 atg ctg ctg cag aat gag ctc ttc ctg aat gtgggc acc aag gac ttc 1776 Met Leu Leu Gln Asn Glu Leu Phe Leu Asn Val GlyThr Lys Asp Phe 495 500 505 510 cca gac gga gag ctt cgg ggg cac gtg gctgcc ctg ccc tac tgt ggg 1824 Pro Asp Gly Glu Leu Arg Gly His Val Ala AlaLeu Pro Tyr Cys Gly 515 520 525 cat agc gcc cgc cat gac acg ctg ccc gtgccc cta gca gga gcc ctg 1872 His Ser Ala Arg His Asp Thr Leu Pro Val ProLeu Ala Gly Ala Leu 530 535 540 gtg cta ccc cct gtg aag agc caa gca gcaggg cac gcc tgg ctt tcc 1920 Val Leu Pro Pro Val Lys Ser Gln Ala Ala GlyHis Ala Trp Leu Ser 545 550 555 ttg gat acc cac tgt cac ctg cac tat gaagtg ctg ctg gct ggg ctt 1968 Leu Asp Thr His Cys His Leu His Tyr Glu ValLeu Leu Ala Gly Leu 560 565 570 ggt ggc tca gaa caa ggc act gtc act gcccac ctc ctt ggg cct cct 2016 Gly Gly Ser Glu Gln Gly Thr Val Thr Ala HisLeu Leu Gly Pro Pro 575 580 585 590 gga acg cca ggg cct cgg cgg ctg ctgaag gga ttc tat ggc tca gag 2064 Gly Thr Pro Gly Pro Arg Arg Leu Leu LysGly Phe Tyr Gly Ser Glu 595 600 605 gcc cag ggt gtg gtg aag gac ctg gagccg gaa ctg ctg cgg cac ctg 2112 Ala Gln Gly Val Val Lys Asp Leu Glu ProGlu Leu Leu Arg His Leu 610 615 620 gca aaa ggc atg gcc tcc ctg ctg atcacc acc aag ggt agc ccc aga 2160 Ala Lys Gly Met Ala Ser Leu Leu Ile ThrThr Lys Gly Ser Pro Arg 625 630 635 ggg gag ctc cga ggg cag gtg cac atagcc aac caa tgt gag gtt ggc 2208 Gly Glu Leu Arg Gly Gln Val His Ile AlaAsn Gln Cys Glu Val Gly 640 645 650 gga ctg cgc ctg gag gcg gcc ggg gccgag ggg gtg cgg gcg ctg ggg 2256 Gly Leu Arg Leu Glu Ala Ala Gly Ala GluGly Val Arg Ala Leu Gly 655 660 665 670 gct ccg gat aca gcc tct gct gcgccg cct gtg gtg cct ggt ctc ccg 2304 Ala Pro Asp Thr Ala Ser Ala Ala ProPro Val Val Pro Gly Leu Pro 675 680 685 gcc cta gcg ccc gcc aaa cct ggtggt cct ggg cgg ccc cga gac ccc 2352 Ala Leu Ala Pro Ala Lys Pro Gly GlyPro Gly Arg Pro Arg Asp Pro 690 695 700 aac aca tgc ttc ttc gag ggg cagcag cgc ccc cac ggg gct cgc tgg 2400 Asn Thr Cys Phe Phe Glu Gly Gln GlnArg Pro His Gly Ala Arg Trp 705 710 715 gcg ccc aac tac gac ccg ctc tgctca ctc tgc acc tgc cag aga cga 2448 Ala Pro Asn Tyr Asp Pro Leu Cys SerLeu Cys Thr Cys Gln Arg Arg 720 725 730 acg gtg atc tgt gac ccg gtg gtgtgc cca ccg ccc agc tgc cca cac 2496 Thr Val Ile Cys Asp Pro Val Val CysPro Pro Pro Ser Cys Pro His 735 740 745 750 ccg gtg cag gct ccc gac cagtgc tgc cct gtt tgc cct gag aaa caa 2544 Pro Val Gln Ala Pro Asp Gln CysCys Pro Val Cys Pro Glu Lys Gln 755 760 765 gat gtc aga gac ttg cca gggctg cca agg agc cgg gac cca gga gag 2592 Asp Val Arg Asp Leu Pro Gly LeuPro Arg Ser Arg Asp Pro Gly Glu 770 775 780 ggc tgc tat ttt gat ggt gaccgg agc tgg cgg gca gcg ggt acg cgg 2640 Gly Cys Tyr Phe Asp Gly Asp ArgSer Trp Arg Ala Ala Gly Thr Arg 785 790 795 tgg cac ccc gtt gtg ccc cccttt ggc tta att aag tgt gct gtc tgc 2688 Trp His Pro Val Val Pro Pro PheGly Leu Ile Lys Cys Ala Val Cys 800 805 810 acc tgc aag ggg ggc act ggagag gtg cac tgt gag aag gtg cag tgt 2736 Thr Cys Lys Gly Gly Thr Gly GluVal His Cys Glu Lys Val Gln Cys 815 820 825 830 ccc cgg ctg gcc tgt gcccag cct gtg cgt gtc aac ccc acc gac tgc 2784 Pro Arg Leu Ala Cys Ala GlnPro Val Arg Val Asn Pro Thr Asp Cys 835 840 845 tgc aaa cag tgt cca gtgggg tcg ggg gcc cac ccc cag ctg ggg gac 2832 Cys Lys Gln Cys Pro Val GlySer Gly Ala His Pro Gln Leu Gly Asp 850 855 860 ccc atg cag gct gat gggccc cgg ggc tgc cgt ttt gct ggg cag tgg 2880 Pro Met Gln Ala Asp Gly ProArg Gly Cys Arg Phe Ala Gly Gln Trp 865 870 875 ttc cca gag agt cag agctgg cac ccc tca gtg ccc cct ttt gga gag 2928 Phe Pro Glu Ser Gln Ser TrpHis Pro Ser Val Pro Pro Phe Gly Glu 880 885 890 atg agc tgt atc acc tgcaga tgt ggg gca ggg gtg cct cac tgt gag 2976 Met Ser Cys Ile Thr Cys ArgCys Gly Ala Gly Val Pro His Cys Glu 895 900 905 910 cgg gat gac tgt tcactg cca ctg tcc tgt ggc tcg ggg aag gag agt 3024 Arg Asp Asp Cys Ser LeuPro Leu Ser Cys Gly Ser Gly Lys Glu Ser 915 920 925 cga tgc tgt tcc cgctgc acg gcc cac cgg cgg cca gcc cca gag acc 3072 Arg Cys Cys Ser Arg CysThr Ala His Arg Arg Pro Ala Pro Glu Thr 930 935 940 aga act gat cca gagctg gag aaa gaa gcc gaa ggc tct tag 3114 Arg Thr Asp Pro Glu Leu Glu LysGlu Ala Glu Gly Ser 945 950 955 ggagcagcca gagggccaag tgaccaagaggatggggcct gagctgggga aggggtggca 3174 tcgaggacct tcttgcattc tcctgtgggaagcccagtgc ctttgctcct ctgtcctgcc 3234 tctactccca cccccactac ctctgggaaccacagctcca caagggggag aggcagctgg 3294 gccagaccga ggtcacagcc actccaagtcctgccctgcc accctcggcc tctgtcctgg 3354 aagccccacc cctttcctcc tgtacataatgtcactggct tgttgggatt tttaatttat 3414 cttcactcag caccaagggc ccccgacactccactcctgc tgcccctgag ctgagcagag 3474 tcattattgg agagttttgt atttattaaaacatttcttt ttcagtcaaa aaaaaaaaaa 3534 aaaaaaaaaa aaa 3547 8 955 PRT Homosapiens 8 Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Leu Leu Leu Leu GlyLeu 1 5 10 15 Leu Leu Leu Gly Ser Arg Pro Ala Arg Gly Ala Gly Pro GluPro Pro 20 25 30 Val Leu Pro Ile Arg Ser Glu Lys Glu Pro Leu Pro Val ArgGly Ala 35 40 45 Ala Gly Cys Thr Phe Gly Gly Lys Val Tyr Ala Leu Asp GluThr Trp 50 55 60 His Pro Asp Leu Gly Glu Pro Phe Gly Val Met Arg Cys ValLeu Cys 65 70 75 80 Ala Cys Glu Ala Pro Gln Trp Gly Arg Arg Thr Arg GlyPro Gly Arg 85 90 95 Val Ser Cys Lys Asn Ile Lys Pro Glu Cys Pro Thr ProAla Cys Gly 100 105 110 Gln Pro Arg Gln Leu Pro Gly His Cys Cys Gln ThrCys Pro Gln Glu 115 120 125 Arg Ser Ser Ser Glu Arg Gln Pro Ser Gly LeuSer Phe Glu Tyr Pro 130 135 140 Arg Asp Pro Glu His Arg Ser Tyr Ser AspArg Gly Glu Pro Gly Ala 145 150 155 160 Glu Glu Arg Ala Arg Gly Asp GlyHis Thr Asp Phe Val Ala Leu Leu 165 170 175 Thr Gly Pro Arg Ser Gln AlaVal Ala Arg Ala Arg Val Ser Leu Leu 180 185 190 Arg Ser Ser Leu Arg PheSer Ile Ser Tyr Arg Arg Leu Asp Arg Pro 195 200 205 Thr Arg Ile Arg PheSer Asp Ser Asn Gly Ser Val Leu Phe Glu His 210 215 220 Pro Ala Ala ProThr Gln Asp Gly Leu Val Cys Gly Val Trp Arg Ala 225 230 235 240 Val ProArg Leu Ser Leu Arg Leu Leu Arg Ala Glu Gln Leu His Val 245 250 255 AlaLeu Val Thr Leu Thr His Pro Ser Gly Glu Val Trp Gly Pro Leu 260 265 270Ile Arg His Arg Ala Leu Ala Ala Glu Thr Phe Ser Ala Ile Leu Thr 275 280285 Leu Glu Gly Pro Pro Gln Gln Gly Val Gly Gly Ile Thr Leu Leu Thr 290295 300 Leu Ser Asp Thr Glu Asp Ser Leu His Phe Leu Leu Leu Phe Arg Gly305 310 315 320 Leu Leu Glu Pro Arg Ser Gly Gly Leu Thr Gln Val Pro LeuArg Leu 325 330 335 Gln Ile Leu His Gln Gly Gln Leu Leu Arg Glu Leu GlnAla Asn Val 340 345 350 Ser Ala Gln Glu Pro Gly Phe Ala Glu Val Leu ProAsn Leu Thr Val 355 360 365 Gln Glu Met Asp Trp Leu Val Leu Gly Glu LeuGln Met Ala Leu Glu 370 375 380 Trp Ala Gly Arg Pro Gly Leu Arg Ile SerGly His Ile Ala Ala Arg 385 390 395 400 Lys Ser Cys Asp Val Leu Gln SerVal Leu Cys Gly Ala Asp Ala Leu 405 410 415 Ile Pro Val Gln Thr Gly AlaAla Gly Ser Ala Ser Leu Thr Leu Leu 420 425 430 Gly Asn Gly Ser Leu IleTyr Gln Val Gln Val Val Gly Thr Ser Ser 435 440 445 Glu Val Val Ala MetThr Leu Glu Thr Lys Pro Gln Arg Arg Asp Gln 450 455 460 Arg Thr Val LeuCys His Met Ala Gly Leu Gln Pro Gly Gly His Thr 465 470 475 480 Ala ValGly Ile Cys Pro Gly Leu Gly Ala Arg Gly Ala His Met Leu 485 490 495 LeuGln Asn Glu Leu Phe Leu Asn Val Gly Thr Lys Asp Phe Pro Asp 500 505 510Gly Glu Leu Arg Gly His Val Ala Ala Leu Pro Tyr Cys Gly His Ser 515 520525 Ala Arg His Asp Thr Leu Pro Val Pro Leu Ala Gly Ala Leu Val Leu 530535 540 Pro Pro Val Lys Ser Gln Ala Ala Gly His Ala Trp Leu Ser Leu Asp545 550 555 560 Thr His Cys His Leu His Tyr Glu Val Leu Leu Ala Gly LeuGly Gly 565 570 575 Ser Glu Gln Gly Thr Val Thr Ala His Leu Leu Gly ProPro Gly Thr 580 585 590 Pro Gly Pro Arg Arg Leu Leu Lys Gly Phe Tyr GlySer Glu Ala Gln 595 600 605 Gly Val Val Lys Asp Leu Glu Pro Glu Leu LeuArg His Leu Ala Lys 610 615 620 Gly Met Ala Ser Leu Leu Ile Thr Thr LysGly Ser Pro Arg Gly Glu 625 630 635 640 Leu Arg Gly Gln Val His Ile AlaAsn Gln Cys Glu Val Gly Gly Leu 645 650 655 Arg Leu Glu Ala Ala Gly AlaGlu Gly Val Arg Ala Leu Gly Ala Pro 660 665 670 Asp Thr Ala Ser Ala AlaPro Pro Val Val Pro Gly Leu Pro Ala Leu 675 680 685 Ala Pro Ala Lys ProGly Gly Pro Gly Arg Pro Arg Asp Pro Asn Thr 690 695 700 Cys Phe Phe GluGly Gln Gln Arg Pro His Gly Ala Arg Trp Ala Pro 705 710 715 720 Asn TyrAsp Pro Leu Cys Ser Leu Cys Thr Cys Gln Arg Arg Thr Val 725 730 735 IleCys Asp Pro Val Val Cys Pro Pro Pro Ser Cys Pro His Pro Val 740 745 750Gln Ala Pro Asp Gln Cys Cys Pro Val Cys Pro Glu Lys Gln Asp Val 755 760765 Arg Asp Leu Pro Gly Leu Pro Arg Ser Arg Asp Pro Gly Glu Gly Cys 770775 780 Tyr Phe Asp Gly Asp Arg Ser Trp Arg Ala Ala Gly Thr Arg Trp His785 790 795 800 Pro Val Val Pro Pro Phe Gly Leu Ile Lys Cys Ala Val CysThr Cys 805 810 815 Lys Gly Gly Thr Gly Glu Val His Cys Glu Lys Val GlnCys Pro Arg 820 825 830 Leu Ala Cys Ala Gln Pro Val Arg Val Asn Pro ThrAsp Cys Cys Lys 835 840 845 Gln Cys Pro Val Gly Ser Gly Ala His Pro GlnLeu Gly Asp Pro Met 850 855 860 Gln Ala Asp Gly Pro Arg Gly Cys Arg PheAla Gly Gln Trp Phe Pro 865 870 875 880 Glu Ser Gln Ser Trp His Pro SerVal Pro Pro Phe Gly Glu Met Ser 885 890 895 Cys Ile Thr Cys Arg Cys GlyAla Gly Val Pro His Cys Glu Arg Asp 900 905 910 Asp Cys Ser Leu Pro LeuSer Cys Gly Ser Gly Lys Glu Ser Arg Cys 915 920 925 Cys Ser Arg Cys ThrAla His Arg Arg Pro Ala Pro Glu Thr Arg Thr 930 935 940 Asp Pro Glu LeuGlu Lys Glu Ala Glu Gly Ser 945 950 955 9 3299 DNA Homo sapiens source(1)..(3299) small intestine 9 ataataatta ggccaagcgt tgaatagtacgggggggggg ggggggcgag ccccggcggc 60 tctggccgcg gccgcactca gcgccacgcgtcgaaagcgc aggccccgag gacccgccgc 120 actgacagt atg agc cgc aca gcc tacacg gtg gga gcc ctg ctt ctc ctc 171 Met Ser Arg Thr Ala Tyr Thr Val GlyAla Leu Leu Leu Leu 1 5 10 ttg ggg acc ctg ctg ccg gct gct gaa ggg aaaaag aaa ggg tcc caa 219 Leu Gly Thr Leu Leu Pro Ala Ala Glu Gly Lys LysLys Gly Ser Gln 15 20 25 30 ggt gcc atc ccc ccg cca gac aag gcc cag cacaat gac tca gag cag 267 Gly Ala Ile Pro Pro Pro Asp Lys Ala Gln His AsnAsp Ser Glu Gln 35 40 45 act cag tcg ccc cag cag cct ggc tcc agg aac cggggg cgg ggc caa 315 Thr Gln Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg GlyArg Gly Gln 50 55 60 ggg cgg ggc act gcc atg ccc ggg gag gag gtg ctg gagtcc agc caa 363 Gly Arg Gly Thr Ala Met Pro Gly Glu Glu Val Leu Glu SerSer Gln 65 70 75 gag gcc ctg cat gtg acg gag cgc aaa tac ctg aag cga gactgg tgc 411 Glu Ala Leu His Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp TrpCys 80 85 90 aaa acc cag ccg ctt aag cag acc atc cac gag gaa ggc tgc aacagt 459 Lys Thr Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn Ser95 100 105 110 cgc acc atc atc aac cgc ttc tgt tac ggc cag tgc aac tctttc tac 507 Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser PheTyr 115 120 125 atc ccc agg cac atc cgg aag gag gaa ggt tcc ttt cag tcctgc tcc 555 Ile Pro Arg His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser CysSer 130 135 140 ttc tgc aag ccc aag aaa ttc act acc atg atg gtc aca ctcaac tgc 603 Phe Cys Lys Pro Lys Lys Phe Thr Thr Met Met Val Thr Leu AsnCys 145 150 155 cct gaa cta cag cca cct acc aag aag aag aga gtc aca cgtgtg aag 651 Pro Glu Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg ValLys 160 165 170 cag tgt cgt tgc ata tcc atc gat ttg gat taa gccaaatccaggtgcaccca 704 Gln Cys Arg Cys Ile Ser Ile Asp Leu Asp 175 180gcatgtccta ggaatgcaga cccaggaagt cccagaccta aaacaaccag attcttactt 764ggcttaaacc tagaggccag aagaaccccc agctgcctcc tggcaggagc ctgcttgtgc 824gtagttcgtg tgcatgagtg tggatgggtg cctgtgggtg tttttagaca ccagagaaaa 884cacagtctct gctagagagc acttcctatt ttgtaaacct atctgcttta atggggatgt 944accagaaacc cacctcaccc cggctcacat ctaaaggggc ggggccgtgg tctggttctg 1004actttgtgtt tttgtgccct cctggggacc agaatctcct ttcggaatga atgttcatgg 1064aagaggctcc tctgagggca agagacctgt tttagtgctg cattcgacat ggaaaagtcc 1124ttttaacctg tgcttgcatc ctcctttcct cctcctcctc acaatccatc tcttcttaag 1184ttgacagtga ctatgtcagt ctaatctctt gtttgccagg gttcctaaat taattcactt 1244aaccatgatg caaatgtttt tcatttggtg aagacctcca gactctggga gaggctggtg 1304tgggcaagga caagcaggat agtggagtga gaaagggagg gtggagggtg aggccaaatc 1364aggtccagca aaagtcagta gggacattgc agaagcttga aaggccaata ccagaacaca 1424ggctgatgct tctgagaaag tcttttccta gtatttaaca aaacccaagt gaacagagga 1484gaaatgagat tgccagaaag tgattaactt tggccgttgc aatctgctca aacctaacac 1544caaactgaaa acataaatac tgaccactcc tatgttcgga cccaagcaag ttagctaaac 1604caaaccaact cctctgcttt gtccctcagg tggaaaagag aggtagttta gaactctctg 1664cataggggtg ggaattaatc aaaaacctca gaggctgaaa ttcctaatac ctttccttta 1724tcgtggttat agtcagctca tttccattcc actatttccc ataatgcttc tgagagccac 1784taacttgatt gataaagatc ctgcctctgc tgagtgtacc tgacagtagt ctaagatgag 1844agagtttagg gactactctg ttttaacaag aaatattttg ggggtctttt tgttttaact 1904attgtcagga gattgggcta aagagaagac gacgagagta aggaaataaa gggaattgcc 1964tctggctaga gagtagttag gtgttaatac ctggtagaga tgtaagggat atgacctccc 2024tttctttatg tgctcacttg aggatctgag gggaccctgt taggagagca tagcatcatg 2084atgtattagc tgttcatctg ctactggttg gatggacata actattgtaa ctattcagta 2144tttactggta ggcactgtcc tctgattaaa cttggcctac tggcaatggc tacttaggat 2204tgatctaagg gccaaagtgc agggtgggtg aactttattg tactttggat ttggttaacc 2264tgttttcctc aagcctgagg ttttatatac aaactccctg aatactcttt ttgccttgtt 2324acttctcagc ctcctagcca agtcctatgt aatatggaaa acaaacactg cagacttgag 2384attcagttgc cgatcaaggc tctggcattc agagaaccct tgcaactcga gaagctgttt 2444ttgatttcgt ttttgttttg aaccggtgct ctcccatcta acaactaaca aggaccattt 2504ccaggcggga gatattttaa acacccaaaa tgttgggtct gatttccaaa cttttaaact 2564cactactgat gattctcacg ctaggcgaat ttgtccaaac acatagtgtg tgtgttttgt 2624atacactgta tgaccccacc ccaaatcttt gtattgtcca cattctccaa caataaagca 2684cagagtggat ttaattaagc acacaaatgc taaggcagaa ttttgagggt gggagagaag 2744aaaagggaaa gaagctgaaa atgtaaaacc acaccaggga ggaaaaatga cattcagaac 2804caccaaacac tgaatttctc ttgttgtttt aactctccca caagaatgca atttcgttaa 2864tggagatgac ttaagttggc agcagtaatc ttcttttagg agcttgtacc acagtcttgc 2924acataagtgc agatttgccc caagtaaaga gaatttcctc aacactaact tcacggggat 2984aatcaccacg taactaccct taaagcatat cactagccaa agaggggaat atctgttctt 3044cttactgtgc ctatattaag actagtacaa atgtggtgtg tcttccaact ttcattgaaa 3104atgccatatc tataccatat tttattcgag tcactgatga tgtaatgata tattttttca 3164ttattatagt agaatatttt tatggcaaga gatttgtggt cttgatcata cctattaaaa 3224taatgccaaa caccaaatat gaattttatg atgtacactt tgtgcttggc attaaaagaa 3284aaaaacacac acgcc 3299 10 184 PRT Homo sapiens 10 Met Ser Arg Thr Ala TyrThr Val Gly Ala Leu Leu Leu Leu Leu Gly 1 5 10 15 Thr Leu Leu Pro AlaAla Glu Gly Lys Lys Lys Gly Ser Gln Gly Ala 20 25 30 Ile Pro Pro Pro AspLys Ala Gln His Asn Asp Ser Glu Gln Thr Gln 35 40 45 Ser Pro Gln Gln ProGly Ser Arg Asn Arg Gly Arg Gly Gln Gly Arg 50 55 60 Gly Thr Ala Met ProGly Glu Glu Val Leu Glu Ser Ser Gln Glu Ala 65 70 75 80 Leu His Val ThrGlu Arg Lys Tyr Leu Lys Arg Asp Trp Cys Lys Thr 85 90 95 Gln Pro Leu LysGln Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr 100 105 110 Ile Ile AsnArg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro 115 120 125 Arg HisIle Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys Ser Phe Cys 130 135 140 LysPro Lys Lys Phe Thr Thr Met Met Val Thr Leu Asn Cys Pro Glu 145 150 155160 Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys 165170 175 Arg Cys Ile Ser Ile Asp Leu Asp 180 11 804 DNA Homo sapienssource (1)..(804) 11 atg cat ctc ctc tta ttt cag ctg ctg gta ctc ctg cctcta gga aag 48 Met His Leu Leu Leu Phe Gln Leu Leu Val Leu Leu Pro LeuGly Lys 1 5 10 15 acc aca cgg cac cag gat ggc cgc cag aat cag agt tctctt tcc ccc 96 Thr Thr Arg His Gln Asp Gly Arg Gln Asn Gln Ser Ser LeuSer Pro 20 25 30 gta ctc ctg cca agg aat caa aga gag ctt ccc aca ggc aaccat gag 144 Val Leu Leu Pro Arg Asn Gln Arg Glu Leu Pro Thr Gly Asn HisGlu 35 40 45 gaa gct gag gag aag cca gat ctg ttt gtc gca gtg cca cac cttgta 192 Glu Ala Glu Glu Lys Pro Asp Leu Phe Val Ala Val Pro His Leu Val50 55 60 gcc acc agc cct gca ggg gaa ggc cag agg cag aga gag aag atg ctg240 Ala Thr Ser Pro Ala Gly Glu Gly Gln Arg Gln Arg Glu Lys Met Leu 6570 75 80 tcc aga ttt ggc agg ttc tgg aag aag cct gag aga gaa atg cat cca288 Ser Arg Phe Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro 8590 95 tcc agg gac tca gat agt gag ccc ttc cca cct ggg acc cag tcc ctc336 Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Gln Ser Leu 100105 110 atc cag ccg ata gat gga atg aaa atg gag aaa tct cct ctt cgg gaa384 Ile Gln Pro Ile Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg Glu 115120 125 gaa gcc aag aaa ttc tgg cac cac ttc atg ttc aga aaa act ccg gct432 Glu Ala Lys Lys Phe Trp His His Phe Met Phe Arg Lys Thr Pro Ala 130135 140 tct cag ggg gtc atc ttg ccc atc aaa agc cat gaa gta cat tgg gag480 Ser Gln Gly Val Ile Leu Pro Ile Lys Ser His Glu Val His Trp Glu 145150 155 160 acc tgc agg aca gtg ccc ttc agc cag act ata acc cac gaa ggctgt 528 Thr Cys Arg Thr Val Pro Phe Ser Gln Thr Ile Thr His Glu Gly Cys165 170 175 gaa aaa gta gtt gtt cag aac aac ctt tgc ttt ggg aaa tgc gggtct 576 Glu Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser180 185 190 gtt cat ttt cct gga gcc gcg cag cac tcc cat acc tcc tgc tctcac 624 Val His Phe Pro Gly Ala Ala Gln His Ser His Thr Ser Cys Ser His195 200 205 tgt ttg cct gcc aag ttc acc acg atg cac ttg cca ctg aac tgcact 672 Cys Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr210 215 220 gaa ctt tcc tcc gtg atc aag gtg gtg atg ctg gtg gag gag tgccag 720 Glu Leu Ser Ser Val Ile Lys Val Val Met Leu Val Glu Glu Cys Gln225 230 235 240 tgc aag gtg aag acg gag cat gaa gat gga cac atc cta catgct ggc 768 Cys Lys Val Lys Thr Glu His Glu Asp Gly His Ile Leu His AlaGly 245 250 255 tcc cag gat tcc ttt atc cca gga gtt tca gct tga 804 SerGln Asp Ser Phe Ile Pro Gly Val Ser Ala 260 265 12 267 PRT Homo sapiensmisc_feature (361)..(741) DAN domain 12 Met His Leu Leu Leu Phe Gln LeuLeu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 Thr Thr Arg His Gln Asp GlyArg Gln Asn Gln Ser Ser Leu Ser Pro 20 25 30 Val Leu Leu Pro Arg Asn GlnArg Glu Leu Pro Thr Gly Asn His Glu 35 40 45 Glu Ala Glu Glu Lys Pro AspLeu Phe Val Ala Val Pro His Leu Val 50 55 60 Ala Thr Ser Pro Ala Gly GluGly Gln Arg Gln Arg Glu Lys Met Leu 65 70 75 80 Ser Arg Phe Gly Arg PheTrp Lys Lys Pro Glu Arg Glu Met His Pro 85 90 95 Ser Arg Asp Ser Asp SerGlu Pro Phe Pro Pro Gly Thr Gln Ser Leu 100 105 110 Ile Gln Pro Ile AspGly Met Lys Met Glu Lys Ser Pro Leu Arg Glu 115 120 125 Glu Ala Lys LysPhe Trp His His Phe Met Phe Arg Lys Thr Pro Ala 130 135 140 Ser Gln GlyVal Ile Leu Pro Ile Lys Ser His Glu Val His Trp Glu 145 150 155 160 ThrCys Arg Thr Val Pro Phe Ser Gln Thr Ile Thr His Glu Gly Cys 165 170 175Glu Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser 180 185190 Val His Phe Pro Gly Ala Ala Gln His Ser His Thr Ser Cys Ser His 195200 205 Cys Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr210 215 220 Glu Leu Ser Ser Val Ile Lys Val Val Met Leu Val Glu Glu CysGln 225 230 235 240 Cys Lys Val Lys Thr Glu His Glu Asp Gly His Ile LeuHis Ala Gly 245 250 255 Ser Gln Asp Ser Phe Ile Pro Gly Val Ser Ala 260265 13 2032 DNA Homo sapiens source (1)..(2032) Homo sapiens Taxon960613 cgcggggcgc ggagtcggcg gggcctcgcg ggacgcgggc agtgcggaga ccgcggcgct 60gaggacgcgg gagccgggag cgcacgcgcg gggtggagtt cagcctactc tttcttagat 120gtgaaaggaa aggaagatca tttcatgcct tgttgataaa ggttcagact tctgctgatt 180cataaccatt tggctctgag ctatgacaag agaggaaaca aaaagttaaa cttacaagcc 240tgccataagt gagaagcaaa cttccttgat aac atg ctt ttg cga agt gca gga 294 MetLeu Leu Arg Ser Ala Gly 1 5 aaa tta aat gtg ggc acc aag aaa gag gat ggtgag agt aca gcc ccc 342 Lys Leu Asn Val Gly Thr Lys Lys Glu Asp Gly GluSer Thr Ala Pro 10 15 20 acc ccc cgt cca aag gtc ttg cgt tgt aaa tgc caccac cat tgt cca 390 Thr Pro Arg Pro Lys Val Leu Arg Cys Lys Cys His HisHis Cys Pro 25 30 35 gaa gac tca gtc aac aat att tgc agc aca gac gga tattgt ttc acg 438 Glu Asp Ser Val Asn Asn Ile Cys Ser Thr Asp Gly Tyr CysPhe Thr 40 45 50 55 atg ata gaa gag gat gac tct ggg ttg cct gtg gtc acttct ggt tgc 486 Met Ile Glu Glu Asp Asp Ser Gly Leu Pro Val Val Thr SerGly Cys 60 65 70 cta gga cta gaa ggc tca gat ttt cag tgt cgg gac act cccatt cct 534 Leu Gly Leu Glu Gly Ser Asp Phe Gln Cys Arg Asp Thr Pro IlePro 75 80 85 cat caa aga aga tca att gaa tgc tgc aca gaa agg aac gaa tgtaat 582 His Gln Arg Arg Ser Ile Glu Cys Cys Thr Glu Arg Asn Glu Cys Asn90 95 100 aaa gac cta cac cct aca ctg cct cca ttg aaa aac aga gat tttgtt 630 Lys Asp Leu His Pro Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Val105 110 115 gat gga cct ata cac cac agg gct tta ctt ata tct gtg act gtctgt 678 Asp Gly Pro Ile His His Arg Ala Leu Leu Ile Ser Val Thr Val Cys120 125 130 135 agt ttg ctc ttg gtc ctt atc ata tta ttt tgt tac ttc cggtat aaa 726 Ser Leu Leu Leu Val Leu Ile Ile Leu Phe Cys Tyr Phe Arg TyrLys 140 145 150 aga caa gaa acc aga cct cga tac agc att ggg tta gaa caggat gaa 774 Arg Gln Glu Thr Arg Pro Arg Tyr Ser Ile Gly Leu Glu Gln AspGlu 155 160 165 act tac att cct cct gga gaa tcc ctg aga gac tta att gagcag tct 822 Thr Tyr Ile Pro Pro Gly Glu Ser Leu Arg Asp Leu Ile Glu GlnSer 170 175 180 cag agc tca gga agt gga tca ggc ctc cct ctg ctg gtc caaagg act 870 Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu Val Gln ArgThr 185 190 195 ata gct aag cag att cag atg gtg aaa cag att gga aaa ggtcgc tat 918 Ile Ala Lys Gln Ile Gln Met Val Lys Gln Ile Gly Lys Gly ArgTyr 200 205 210 215 ggg gaa gtt tgg atg gga aag tgg cgt ggc gaa aag gtagct gtg aaa 966 Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu Lys Val AlaVal Lys 220 225 230 gtg ttc ttc acc aca gag gaa gcc agc tgg ttc aga gagaca gaa ata 1014 Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe Arg Glu ThrGlu Ile 235 240 245 tat cag aca gtg ttg atg agg cat gaa aac att ttg ggtttc att gct 1062 Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile Leu Gly PheIle Ala 250 255 260 gca gat atc aaa ggg aca ggg tcc tgg acc cag ttg taccta atc aca 1110 Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln Leu Tyr LeuIle Thr 265 270 275 gac tat cat gaa aat ggt tcc ctt tat gat tat ctg aagtcc acc acc 1158 Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Tyr Leu Lys SerThr Thr 280 285 290 295 cta gac gct aaa tca atg ctg aag tta gcc tac tcttct gtc agt ggc 1206 Leu Asp Ala Lys Ser Met Leu Lys Leu Ala Tyr Ser SerVal Ser Gly 300 305 310 tta tgt cat tta cac aca gaa atc ttt agt act caaggc aaa cca gca 1254 Leu Cys His Leu His Thr Glu Ile Phe Ser Thr Gln GlyLys Pro Ala 315 320 325 att gcc cat cga gat ctg aaa agt aaa aac att ctggtg aag aaa aat 1302 Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile Leu ValLys Lys Asn 330 335 340 gga act tgc tgt att gct gac ctg ggc ctg gct gttaaa ttt att agt 1350 Gly Thr Cys Cys Ile Ala Asp Leu Gly Leu Ala Val LysPhe Ile Ser 345 350 355 gat aca aat gaa gtt gac ata cca cct aac act cgagtt ggc acc aaa 1398 Asp Thr Asn Glu Val Asp Ile Pro Pro Asn Thr Arg ValGly Thr Lys 360 365 370 375 cgc tat atg cct cca gaa gtg ttg gac gag agcttg aac aga aat cac 1446 Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser LeuAsn Arg Asn His 380 385 390 ttc cag tct tac atc atg gct gac atg tat agtttt ggc ctc atc ctt 1494 Phe Gln Ser Tyr Ile Met Ala Asp Met Tyr Ser PheGly Leu Ile Leu 395 400 405 tgg gag gtt gct agg aga tgt gta tca gga ggtata gtg gaa gaa tac 1542 Trp Glu Val Ala Arg Arg Cys Val Ser Gly Gly IleVal Glu Glu Tyr 410 415 420 cag ctt cct tat cat gac cta gtg ccc agt gacccc tct tat gag gac 1590 Gln Leu Pro Tyr His Asp Leu Val Pro Ser Asp ProSer Tyr Glu Asp 425 430 435 atg agg gag att gtg tgc atc aag aag tta cgcccc tca ttc cca aac 1638 Met Arg Glu Ile Val Cys Ile Lys Lys Leu Arg ProSer Phe Pro Asn 440 445 450 455 cgg tgg agc agt gat gag tgt cta agg cagatg gga aaa ctc atg aca 1686 Arg Trp Ser Ser Asp Glu Cys Leu Arg Gln MetGly Lys Leu Met Thr 460 465 470 gaa tgc tgg gct cac aat cct gca tca aggctg aca gcc ctg cgg gtt 1734 Glu Cys Trp Ala His Asn Pro Ala Ser Arg LeuThr Ala Leu Arg Val 475 480 485 aag aaa aca ctt gcc aaa atg tca gag tcccag gac att aaa ctc tga 1782 Lys Lys Thr Leu Ala Lys Met Ser Glu Ser GlnAsp Ile Lys Leu 490 495 500 taggagagga aaagtaagca tctctgcaga aagccaacaggtactcttct gtttgtgggc 1842 agagcaaaag acatcaaata agcatccaca gtacaagccttgaacatcgt cctgcttccc 1902 agtgggttca gacctcacct ttcagggagc gacctgggcaaagacagaga agctcccaga 1962 aggagagatt gatccgtgtc tgtttgtagg cggagaaaccgttgggtaac ttgttcaaga 2022 tatgatgcat 2032 14 502 PRT Homo sapiensmisc_feature (367)..(606) Activin_recp; Region Activin types I and II 14Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys Glu 1 5 1015 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu Arg Cys 20 2530 Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn Ile Cys Ser 35 4045 Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu Asp Asp Ser Gly Leu 50 5560 Pro Val Val Thr Ser Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 7075 80 Cys Arg Asp Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 8590 95 Thr Glu Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro100 105 110 Leu Lys Asn Arg Asp Phe Val Asp Gly Pro Ile His His Arg AlaLeu 115 120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu IleIle Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg ProArg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile ProPro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser Gln Ser SerGly Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln Arg Thr Ile AlaLys Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly Lys Gly Arg Tyr GlyGlu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly Glu Lys Val Ala Val LysVal Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235 240 Trp Phe Arg Glu ThrGlu Ile Tyr Gln Thr Val Leu Met Arg His Glu 245 250 255 Asn Ile Leu GlyPhe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp 260 265 270 Thr Gln LeuTyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr 275 280 285 Asp TyrLeu Lys Ser Thr Thr Leu Asp Ala Lys Ser Met Leu Lys Leu 290 295 300 AlaTyr Ser Ser Val Ser Gly Leu Cys His Leu His Thr Glu Ile Phe 305 310 315320 Ser Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325330 335 Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly340 345 350 Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile ProPro 355 360 365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu ValLeu Asp 370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile MetAla Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Val AlaArg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln Leu ProTyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu Asp Met ArgGlu Ile Val Cys Ile Lys Lys 435 440 445 Leu Arg Pro Ser Phe Pro Asn ArgTrp Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met Gly Lys Leu Met ThrGlu Cys Trp Ala His Asn Pro Ala Ser 465 470 475 480 Arg Leu Thr Ala LeuArg Val Lys Lys Thr Leu Ala Lys Met Ser Glu 485 490 495 Ser Gln Asp IleLys Leu 500 15 20 DNA Homo sapiens 15 acgagagctc tcactggtcc 20 16 20 DNAHomo sapiens 16 cattccggat tacatgaggg 20 17 11233 DNA Homo sapiensmisc_feature (1)..(3165) 5′ flanking region 17 gaattccttc cgtagcttcaccagacacct aattggccaa gaaggtttga agacctgatg 60 tggttcttaa ttggggatggggaattaagg gctactgtat ctataggatt atcttttcac 120 ttgcatagac ctatttggtgtgttcagggc atagtgatac tataattgcc atatttaaca 180 gtttataaag ttcaagcccagcatattctt tgcctgttta atgatgtctt ggtatcagcc 240 ttttaatggt acttatcagcatagaaaatg gaaacaaaat aacttttaaa acagtagctc 300 tcaagcttta gtgtgctcagaatgaccaga gaaccttgtg aaatatacag atttctgggt 360 ccagatctgg ggcaggaccaggaagtctgc atttcatctg cacccccacc ctactctgag 420 gcttatagtc ctgagaacatgctttgaaaa aggctgtccc aagggctcgc agacaggcta 480 ttgaccagct actctttcttgatgttctcc aggaaaaacc caacaaagga atgcctttca 540 ttgagtagta gcagcataggagcaatagtt gctcctgaat tatggtgggt ttcccctctt 600 catcaatgtg ctttaagggtacagtttcat ttggtctatc taccatgttc tataaaaaca 660 tgaaaattca caggtaagtttgagatacag aaaataacta aactgattct tctcacgaac 720 tctgatcact aggctgtggttgatttagct ctctaaccaa caagtaattt gttctttggc 780 atgagtaagg ggggaaaaggaggagtgggt aaaagcagct gataacagat ggcttgcgcc 840 catctaaaat gtggggagagaaataaagct gtcccaagag aactaaagct gagttctctc 900 gtcatatatc tgaagattcatatcaggggt ctaaacatgg tatgtcgggt agcttaattg 960 gaaactcctg gactgtgagtgtcacagact catggatggg ccaatcagtg gccactttag 1020 tgtctgggct gcagcaaaatgagacaatag ctgtcattca caaacctttg gaattaaaaa 1080 aaccccgaaa tgacattggtgctttaaagt aaaataaagt cctgccttta agtccagcat 1140 atcactgttg tttctgagtttaaatattaa gaaccacatt tcgttaatga ttaaaacaac 1200 agtgattgat ttaggggctcagtgagcatt taatctgtcc tgacttcagg taccatgcta 1260 aaggagcaca atgcctgatgctgcaggaga aacattaggt aactatttaa tggagtttta 1320 attttctgtt attatttttaataattaatt gtgattttga ctatttggaa gctacaggta 1380 tattttgtcc tccttttggggtggtgttat tgccctgccc tgttttaatc agtggttctt 1440 agagaaagtg aactcaggagtgacttaaaa tgaaggaaga cggactttgg ctaaaattac 1500 aattaaataa tcaaatcattttcaaatata aagggagcat gcagatgatc tggcccaatc 1560 ctttcattct gcagatgagaaaactgagac tcataggaat gaaaagactt gcccaaagcc 1620 atacagcttg tttctgttgtttggtgcatt aggccaaaag acctaggcct aatagatgga 1680 aaagatggca ggatgtcttggccttgctct gacagttgct tctctgatct cagatatttc 1740 ccaccctttg taatctgtgttccacacagg aagtagttct tgttttttaa atatcgaagg 1800 tgtataaacg taaagtttttatagatgagc cacccagggc caatatctgt ttaagtaaag 1860 acctaaatgc tttgcagagacagtaaagtg tcatgtctgt cccagggaaa gaaatccagg 1920 acaggaaatg ctcagtcttccagcactcct ctggctacct ggagctcagg ctatgagcct 1980 caacccctcc ctgaagcattagctctggag cagaggctgt gatttacttc agagatctgg 2040 gcaagtccct ttaacctggtagtccttcct ttccttgttt gtaaaacaga gagatgaggc 2100 tgatagctcc ctcacagctccatcagaggc agtgtgtgaa attagttcct gtttgggaag 2160 gtttaaaagc caccacattccacctccctg ctaatatgat tactaaaatg tttttatatg 2220 aaagggccaa ttcctcatctcccctcttcc tttaaaaaca gaccaagggg catcttttct 2280 tgtctccctg tggcctaaaaggttactgct tctgtggtta tctccttgga aagacagagt 2340 gtcaggactc ttaggtacaccaaaaatgaa caaaaaaatc aacaacaacc ataacaccaa 2400 caaaaataac tgctgtgtcggttcttaaga cggcttctga gctagaaaca gatttttcta 2460 actgtaaaaa acgtggccccagcctgtctg caggccacct ctgtctttag gccttggggg 2520 gaggagggaa gtgagctcatttactggggt ctacctcagg gtcatcacca aggtgttcta 2580 caaaacgcac tttaagaatgttttggaagg aaattcacct tttaacagcc caagaggtat 2640 ctctctctgg cacacagttctgcacacagc ctgtttctca acgtttggaa atcttttaac 2700 agtttatgga aggccaccttttaaaccgat ccaacagctc ctttctccat aacctgattt 2760 tagaggtgtt tcattatctctaattactca gggtaaatgg tgattactca gtgttttaat 2820 catcagtttg ggcagcagttacactaaact cagggaagcc cagactccca tgggtatttt 2880 tggaaggtac ggcgactagtcggtgcatgc tttctagtac ctccgcacgt ggtccccagg 2940 tgagccccag ccgcttcccagagctggagg cagcggcgtc ccagctccga cggcagctgc 3000 ggactcgggc gctgcctgggcttccgggac ccgggcctgc taggcgaggt cgggcggctg 3060 gaggggagga tgtgggcggggctcccatcc ccagaaaggg aggcgagcga gggaggaggg 3120 aaggagggag gggccgccggggaagaggag gaggaaggaa agaaa gaa agc gag gga 3177 Glu Ser Glu Gly 1 gggaaa gag gag gaa gga aga tgc gag aag gca gag gag gag gga ggg 3225 Gly LysGlu Glu Glu Gly Arg Cys Glu Lys Ala Glu Glu Glu Gly Gly 5 10 15 20 agggaa gga gcg cgg agc ccg gcc cgg aag cta ggt gag tgt ggc atc 3273 Arg GluGly Ala Arg Ser Pro Ala Arg Lys Leu Gly Glu Cys Gly Ile 25 30 35 cga gctgag gga cgc gag cct gag acg ccg ctg ctg ctc cgg ctg agt 3321 Arg Ala GluGly Arg Glu Pro Glu Thr Pro Leu Leu Leu Arg Leu Ser 40 45 50 atc tag cttgtc tcc ccg atg gga ttc ccg tcc aag cta tct cga gcc 3369 Ile Leu Val SerPro Met Gly Phe Pro Ser Lys Leu Ser Arg Ala 55 60 65 tgc agc gcc aca gtcccc ggc cct cgc cca ggt tca ctg caa ccg ttc 3417 Cys Ser Ala Thr Val ProGly Pro Arg Pro Gly Ser Leu Gln Pro Phe 70 75 80 aga ggt ccc cag gag ctgctg ctg gcg agc ccg cta ctg cag gga cct 3465 Arg Gly Pro Gln Glu Leu LeuLeu Ala Ser Pro Leu Leu Gln Gly Pro 85 90 95 atg gtgagcaagg ctacctggtgaggggagaca ggcagagggg gtctaggagc 3518 Met 100 ctccttgggg ggaagaagctggtcacaggc tgtgaccgag gcaaaaggtg gcctaattat 3578 tttccaatag tggtgctggaggtggggatg ctggcgctga aagaccttta aatatcggct 3638 actgcccctg cccaggccttctctgtccag cagtccctgg gagattctca cctttgggaa 3698 gtgcggggca ggagagcagaaacaagagaa gcccttggta ggggggtcgt tgggaaaaac 3758 tgtggggtct tgggctgaacgcgttgccca cgggctggag gttgcgatcc ccggacggaa 3818 agcgcgggag gaggaaggagagaaccggct ctgaggtcca gagagagtga gggggcagag 3878 cgacggcgag atggggagagaacacctagc tggagcaggt tctgcggtag agagcgcagt 3938 cctgctggcc tctggagagtgcgcgccgct acggaggctg cgtcgagggg agtgtcaccc 3998 aatctggccc ccagctggcggggcgccctg agagcttgcg aactgcagtt gcaggacgcg 4058 ccttctccac gagctattttcgtcgacttg cggaacccaa ggaacctcgc ctctatcatt 4118 tcacggtgta gggtccctagagacgacagc caagatccca ggggctccca ggacgcttgt 4178 tcctgcggtg tcgtgtcctatggggagttc ctggcgggac gaaaggcgga cgcgcggctc 4238 ttcctggccc tccaggcccggaaccgacgg gaaaggttcc cgtgattccc gagtccctgc 4298 aggcttcttc cagcgggagttggtccgggg gccttagagg cctccaagca ctgctttgga 4358 ggatggtttc caaggatcgcggtttgtgag ttgaaggctt tgtgagaggt taaaccccca 4418 aaagatacat acttggtaaactgaggctac ctgtaaacac atttcggcat taggagaaga 4478 ttcgagtagg gaagtgaaggacaaccaccc cgagttacat tcctttcccc caataaaaag 4538 ctctggggat gaaagttcttttggctttta tcttttcgat ttaaaaattt gagaagaaaa 4598 atgtgactag agatgaatcctggtgaatcc gaaattgaaa cacaactccc ccttcccctt 4658 cctatcctct cggttttagaaccgcgctct cccgccccag gagattcctt ggggccgagg 4718 gttttccggg gaacccgggcgcccgcccct tctactgtcc ctttgccccg cgggcacagc 4778 ttgcctccgt ctgctttctctacttctgga cctctcctcg ccgggctttt taaagggctt 4838 ctgcgtctca aaacaaaacaaaaaaaccct ttgctcttcc caaccctttc gcagcccgcc 4898 ccagcggtgg cgcgggaccagcaaaggcga aagccgcgcg gctcttgccg ggcgcggacg 4958 gtcgcgcagg ggcgcccgcggcctccgcac ccggacctga ggtgttggtc gactccgggc 5018 atccacggtc gggagggagggctgagctgt tcgatccttt acttttcttc ctcaaagtct 5078 acctgccaat gcccctaagaagaaaaccaa gtatgtgcgt ggagagtggg gcggcaggca 5138 acccgagttc ttgagctccggagcgaccca aagcagcaac tgggaacagc ctcaggaaag 5198 ggaggtcggg tggagtgggctttggggcag gagtcatggg gcccgggccc cggggacgac 5258 ctggcgctcc cggccctgctgaacgctgag ttgcgcctag tcgggttttc gaagaggccc 5318 ttgcgcagag cgacccacgcgcgcggcagc atcttcgatt agtcaggaca tcccagtaac 5378 tgcttgaact gtaggtaggtaaaattcttg aaggagtatt tgctgcgtgc gactctgctg 5438 ctggtgcaac ggaggaagggggtgggggaa ggaagtggcg ggggaaggag tgtggtggtg 5498 gtttaaaaaa taagggaagccgaggcgaga gagacgcaga cgcagaggtc gagcgcaggc 5558 cgaaagctgt tcaccgttttctcgactccg gggaacatgg tgggatttcc tttctgcgcc 5618 gggtcgggag ttgtaaaacctcggccacat taagatctga aaactgtgat gcgtcctttc 5678 tgcagagacg cctctttctgaatctgcccg gagcttcgag ccccggcgtc tgtccctcag 5738 cctggcatgg cttcttcgggggtctgcttt gcatggggag aggggccacg cagcggcgga 5798 ctaggtttgg ggattctcggtaatggaccc ggagcaatga ctaacagccg ctccctctca 5858 ctttcccaca gcgatcaccctctaacaccc tccctcccat tcccggcccc gcgcgtgaca 5918 aggtcggctg ctttcagccgggagctagat cggtggcccg gctcttcgga gccttagcag 5978 gcgttcgcca aggggtgactggctgtcatt gggagcaata tttggccttg aggagaccct 6038 ggggaggaag tggcggggagctcgtgtttg cttgtgtgtg tgtggggggg ggggtgtgtg 6098 tacacgcgcg tgggcagggtccctctgcgc tttccttttt aagtgcctct cggtggtgag 6158 gctttgggcg ggtgagactttcccgacctc gctcccggcc ccacttaagc cgggttcgag 6218 ctgggagacg cagtcccttcagtgcgcccc aaatcctctg gcttcaggtg gcccggcgcg 6278 ggggcccagc acgacgcaccgcgccgagaa ccgggttctc cgtgcgctgc gccagtagcc 6338 ctgggagcgc ggcggccgcggggcaccggc cgagggctct gccgagcgcc gccgggagct 6398 cctcccggac cgctgaggctcgggcggcgg gcgcggaggt tggcctcgcc tggaggggcg 6458 ggcccgcgag gggcggggggctgtggagga ggggagggcg cgcaggccct ttcgccgcct 6518 gccgcgggag gggcctcggcgctcacgtga ctccgagggg ctggaagaaa aacagagcct 6578 gtctgcggtg gagtctcattatattcaaat attcctttta g gag cca ttc cgt agt 6634 Glu Pro Phe Arg Ser 105gcc atc ccg agc aac gca ctg ctg cag ctt ccc tga gcc ttt cca gca 6682 AlaIle Pro Ser Asn Ala Leu Leu Gln Leu Pro Ala Phe Pro Ala 110 115 120 agtttg ttc aag att ggc tgt caa gaa tca tgg act gtt att ata tgc 6730 Ser LeuPhe Lys Ile Gly Cys Gln Glu Ser Trp Thr Val Ile Ile Cys 125 130 135 cttgtt ttc tgt ca gtgagtagac acctcttcct tccccctctc cggaattcac 6784 Leu ValPhe Cys 140 tctgccctca ccacccctgc tcgccggctg tcccttccgt cggacctcctttacaatatc 6844 cacactctgc tccctggcag cactgtcgct cccttcttgg cccggcagccggggcgctgg 6904 aagcgtacgg gttcctttta aagtgctgct agcgcgcact cgccctctcagcgttgcaag 6964 aaaggggagc gcgagggagc taaagagatg aaagcccggg gttgtaccttgagggctaac 7024 cactcccttc ccctatccaa cttgtctggg agagccccca gtgtctccgtggcgcgttcc 7084 cactctcttg tcaaaactca cagaggtctc tccggaatcg tctctcaccccttccctggg 7144 gatgagcggg cacgatcagg cacttttggc tgaatatttc aaactcatcggccacaataa 7204 aataagccct caagccaccc ggttagctcc cagaccacct tctcggcttctggaccctgt 7264 cgccctctgt cttcgcccag cccctgcctc tcactttccc tccctctggctctgaaccaa 7324 ctggaagttg tgaaagttgg gctctgaggg tggaggaaaa gggagagaagctgaaggtct 7384 aaagtggaga gcaatgccat tttaattctc cctcccccac cccttttcaccccctcaatg 7444 ttaactgttt atccttcaag aagccacgct gagatcatgg cccagatagcagttaggaca 7504 aaaaaagatt aacaggatgg aggctatctg atttggggtt atttgactgtaaacaagtta 7564 gaccaagtaa ttacagggca attcttactt tcaggccgtg catggctgcagctggtgggt 7624 gggcgggtgg tgtgagggag aagacacaaa cttgatcttt ctgacctgctttccatcttg 7684 cccctccatt tctagcccta aatgcatatg cagacacatc tctatttctccctatttatt 7744 ggtgtttgtt tattctttaa ccttccactc ccctccccct ccccagagacaccatgattc 7804 ctggtaaccg aatgctgatg gtcgttttat tatgccaagt cctgctaggaggcgcgagcc 7864 atgctagttt gatacctgag acggggaaga aaaaagtcgc cgagattcagggccacgcgg 7924 gaggacgccg ctcagggcag agccatgagc tcctgcggga cttcgaggcgacacttctgc 7984 agatgtttgg gctgcgccgc cgcccgcagc ctagcaagag tgccgtcattccggactaca 8044 tgcgggatct ttaccggctt cagtctgggg aggaggagga agagcagatccacagcactg 8104 gtcttgagta tcctgagcgc ccggccagcc gggccaacac cgtgaggagcttccaccacg 8164 aaggtcagtc tcttccccca gtctgcgtgg gggagggctg gtgggactggctagaggggc 8224 agtgaaagcc ctggggaaga agagttcggg ttacatcaaa ccccagtccaggaggctgag 8284 gaacagagct gcttacctcc aagaatttgc agagctgccg ccgaacttattttttggaga 8344 cagaggggga ggtgttcagg ggaaggggaa tgacagcact cagacgtgggctagccccag 8404 cggtgtgttt ttgctatatc aaagcctttt ctgctaggtt ttctgcccgtttttttcaaa 8464 gcacctactg aatttaatat tacagctgtg tgtttgtcgg gtttattcaataggggcctt 8524 gtaatccgat ctgaatgttt cctagcggat gtttcttttc caaagtaaatctgagttatt 8584 aatccaccag catcattact gtgttggaat ttattttccc ctctgtaacatgatcaacaa 8644 ggcatgctct gtgtttccaa gatcgctggg gaaatgttta gtaacatactcaatagtgga 8704 agagggagag ggtggttgtc tccatgtttc ctcctgcctg tgctctgttggcccctcttt 8764 ttctttacaa ccacttgtaa agaaaactgt ggacacaaag ccaaggtggggggtttaaaa 8824 gaggagtctg attgtggtgc catagaggag ttgacacata gaaattattagacatatcaa 8884 ggaggctgga tatagtttct gtctttggtg cttgagaaat gctagctacattttgctggt 8944 ttgttagctg ccccacttat ctgctccttc aaattaaggg gtatgcttattttcccccag 9004 taggtttccc ctgcataagc agaattcacc attcattgcc caaccctgagctatctcttg 9064 actcttccat ctttgaaaaa agttcatatg ctttttcttt tccccttccttcctaactgt 9124 gcctagaaca tctggagaac atcccaggga ccagtgaaaa ctctgcttttcgtttcctct 9184 ttaacctcag cagcatccct gagaacgagg cgatctcctc tgcagagcttcggctcttcc 9244 gggagcaggt ggaccagggc cctgattggg aaaggggctt ccaccgtataaacatttatg 9304 aggttatgaa gcccccagca gaagtggtgc ctgggcacct catcacacgactactggaca 9364 cgagactggt ccaccacaat gtgacacggt gggaaacttt tgatgtgagccctgcggtcc 9424 ttcgctggac ccgggagaag cagccaaact atgggctagc cattgaggtgactcacctcc 9484 atcagactcg gacccaccag ggccagcatg tcaggattag ccgatcgttacctcaaggga 9544 gtgggaattg ggcccagctc cggcccctcc tggtcacctt tggccatgatggccggggcc 9604 atgccttgac ccgacgccgg agggccaagc gtagccctaa gcatcactcacagcgggcca 9664 ggaagaagaa taagaactgc cggcgccact cgctctatgt ggacttcagcgatgtgggct 9724 ggaatgactg gattgtggcc ccaccaggct accaggcctt ctactgccatggggactgcc 9784 cctttccact ggctgaccac ctcaactcaa ccaaccatgc cattgtgcagaccctggtca 9844 attctgtcaa ttccagtatc cccaaagcct gttgtgtgcc cactgaactgagtgccatct 9904 ccatgctgta cctggatgag tatgataagg tggtactgaa aaattatcaggagatggtag 9964 tagagggatg tgggtgccgc tgagatcagg cagtccttga ggatagacagatatacacac 10024 cacacacaca caccacatac accacacaca cacgttccca tccactcacccacacactac 10084 acagactgct tccttatagc tggactttta tttaaaaaaa aaaaaaaaaaaatggaaaaa 10144 atccctaaac attcaccttg accttattta tgactttacg tgcaaatgttttgaccatat 10204 tgatcatata ttttgacaaa atatatttat aactacgtat taaaagaaaaaaataaaatg 10264 agtcattatt ttaaaggtaa atcatgattt ttttttctcc ttaatcctttctcttttcct 10324 tcgggctcat ctcttttgaa tgaggctttt ttctgttcag gtgagttggaggctggatgg 10384 aagtcaaaag gtggtacctg gaggtggtta agttgtaggg acaggaagtaaactgttggc 10444 agagagagat ggtaattgcc agcatgaatt gttttctatt tctatttaatgttaacaagg 10504 atgcagtatc ctctcccatc tggatgacac atgccttgga gaaacactgggatgaaagga 10564 gtgtaggtca gattaaagac ttcatttcag gccccttgta catcttctgtttcactcacc 10624 tgttgaggtg tatcacagct gagcgtgatg aggtctcaac cctagaaaaatgatacccac 10684 ctctgctttc atgatacctc agggtatctc cagttattac aggtaccaatgtgatatttc 10744 caaatcaaaa ctaatttgta cactaacatc ataatgtgtg tgtgaaggcatgtttttaaa 10804 cttatttttt ttttctccag gtaggactct tttgtttttt cttttgtctttttttttttg 10864 aaacaagttc tctctttgtt gccccaggct ggtcttgaac tcctgggctcaagcaatctt 10924 ctcatttcgg cctctttggg attacaggca tgcactgcta ttttgtcttttttttttttt 10984 tgtaacaaat aatgtaccct accttcaaaa agtttgatga ctactgttttaatatgccac 11044 ttgatagaat ttcccattgt ttcttgactt tttcccttgt cctcttttcccaatgtgaag 11104 gccttcatca agtttaggat cccaacagat tgggctgggt gggggttgacaatggggtca 11164 gatactaaag ggtcagaatt tctaagcagg cactgtgaag gtgtcccactattatacaga 11224 aatctcgag 11233 18 408 PRT Homo sapiens 18 Met Ile ProGly Asn Arg Met Leu Met Val Val Leu Leu Cys Gln Val 1 5 10 15 Leu LeuGly Gly Ala Ser His Ala Ser Leu Ile Pro Glu Thr Gly Lys 20 25 30 Lys LysVal Ala Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly 35 40 45 Gln SerHis Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 50 55 60 Phe GlyLeu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro 65 70 75 80 AspTyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu Glu Glu 85 90 95 GluGln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala Ser 100 105 110Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu His Leu Glu Asn 115 120125 Ile Pro Gly Thr Ser Glu Asn Ser Ala Phe Arg Phe Leu Phe Asn Leu 130135 140 Ser Ser Ile Pro Glu Asn Glu Ala Ile Ser Ser Ala Glu Leu Arg Leu145 150 155 160 Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Trp Glu Arg GlyPhe His 165 170 175 Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala GluVal Val Pro 180 185 190 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg LeuVal His His Asn 195 200 205 Val Thr Arg Trp Glu Thr Phe Asp Val Ser ProAla Val Leu Arg Trp 210 215 220 Thr Arg Glu Lys Gln Pro Asn Tyr Gly LeuAla Ile Glu Val Thr His 225 230 235 240 Leu His Gln Thr Arg Thr His GlnGly Gln His Val Arg Ile Ser Arg 245 250 255 Ser Leu Pro Gln Gly Ser GlyAsn Trp Ala Gln Leu Arg Pro Leu Leu 260 265 270 Val Thr Phe Gly His AspGly Arg Gly His Ala Leu Thr Arg Arg Arg 275 280 285 Arg Ala Lys Arg SerPro Lys His His Ser Gln Arg Ala Arg Lys Lys 290 295 300 Asn Lys Asn CysArg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val 305 310 315 320 Gly TrpAsn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr 325 330 335 CysHis Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr 340 345 350Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile 355 360365 Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu 370375 380 Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met385 390 395 400 Val Val Glu Gly Cys Gly Cys Arg 405

1. A method for the treatment of cancer comprising administering to apatient a therapeutically effective amount of a bone morphogeneticprotein-4 activity inhibitor.
 2. The method of claim 1 wherein the bonemorphogenetic protein-4 activity inhibitor is a polypeptide that bindsspecifically to bone morphogenetic protein-4.
 3. The method of claim 1wherein the bone morphogenetic protein-4 activity inhibitor is apolypeptide that binds specifically to a bone morphogenetic protein-4receptor.
 4. The method of claim 3 wherein the bone morphogeneticprotein-4 receptor is a bone imorphogenetic protein IB receptor.
 5. Themethod of claim 1 wherein the bone morphogenetic protein-4 activityinhibitor is selected from the group consisting of noggin, chordin,cerberus 1 homolog, and gremlin.
 6. The method of claim 1 wherein thebone morphogenetic protein-4 activity inhibitor is noggin.
 7. The methodof claim 6 wherein the amino acid sequence of noggin is selected fromthe group consisting of amino acids #20-231 of SEQ ID NO: 4 and aminoacids #20-231 of SEQ ID No:
 6. 8. The method of claim 1 wherein the bonemorphogenetic protein-4 activity inhibitor is a polypeptide, the aminoacid sequence of which comprises at least ten consecutive amino acids ofa protein selected from the group consisting of noggin, chordin,gremlin, and cerberus 1 homolog
 9. The method of claim 1 wherein thebone morphogenetic protein-4 activity inhibitor is a polypeptide theamino acid sequence of which comprises at least ten consecutive aminoacids of noggin.
 10. The method of claim 9 wherein the amino acidsequence of noggin is selected from the group consisting of SEQ ID NO: 4and SEQ ID NO:
 6. 11. The method of claim 1 wherein the bonemorphogenetic protein-4 activity inhibitor is an antibody to bonemorphogenetic protein-4.
 12. The method of claim 1 wherein the bonemorphogenetic protein-4 activity inhibitor is an antisenseoligonucleotide that binds to a bone morphogenetic protein-4 nucleicacid sequence.
 13. The method of claim 1 wherein the bone morphogeneticprotein-4 activity inhibitor is an antisense oligonucleotide that bindsto at least a portion of a bone morphogenetic protein-4 nucleic acidsequence.
 14. The method of claim 1 wherein the cancer is a carcinoma.15. The method of claim 14 wherein the carcinoma is selected from thegroup consisting of bladder cancer, breast cancer, colon cancer, kidneycancer, lung cancer, ovarian cancer, thyroid cancer, endometrial cancer,omental cancer, testicular cancer, and liver cancer.
 16. The method ofclaim 1 wherein the cancer is lung cancer.
 17. The method of claim 1wherein the patient is a human.
 18. The method of claim 1 wherein thebone morphogenetic protein-4 activity inhibitor further comprises apharmaceutically acceptable carrier.
 19. The method of claim 18 whereinthe bone morphogenetic protein-4 activity inhibitor is administeredorally, enterically, intravenously, peritoneally, subcutaneously,transdermally, parenterally, intratumorally, or rectally.
 20. A methodfor the treatment of cancer comprising administering to a patient atherapeutically effective amount of an expression vector having anucleic acid sequence encoding a bone morphogenetic protein-4 activityinhibitor.
 21. The method of claim 20 wherein the expression vectorfurther comprises a selective promoter that is operably linked to thenucleic acid sequence encoding a bone morphogenetic protein-4 activityinhibitor.
 22. The method of claim 21 wherein the selective promoter iscarcinoembryonic antigen (CEA) promoter.
 23. The method of claim 20wherein the bone morphogenetic protein-4 activity inhibitor is apolypeptide that specifically binds to bone morphogenetic protein-4. 24.The method of claim 20 wherein the bone morphogenetic protein-4 activityinhibitor is a polypeptide that specifically binds to a bonemorphogenetic protein-4 receptor.
 25. The method of claim 24 wherein thebone morphogenetic protein-4 receptor is bone morphogenetic protein IBreceptor.
 26. The method of claim 20 wherein the bone morphogeneticprotein-4 activity inhibitor is selected from the group consisting ofnoggin, chordin, gremlin, and cerberus 1 homolog.
 27. The method ofclaim 20 wherein the bone morphogenetic protein-4 activity inhibitor isnoggin.
 28. The method of claim 27 wherein the amino acid sequence ofnoggin is selected from the group consisting of SEQ ID NO: 4 and SEQ IDNO:
 6. 29. The method of claim 20, wherein the bone morphogeneticprotein-4 activity inhibitor is a polypeptide the amino acid sequence ofwhich comprises at least ten consecutive amino acids of noggin.
 30. Themethod of claim 20, wherein the amino acid sequence of noggin isselected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:
 6. 31.The method of claim 20 wherein the cancer is a carcinoma.
 32. The methodof claim 31 wherein the carcinoma is selected from the group consistingof bladder cancer, breast cancer, colon cancer, kidney cancer, lungcancer, ovarian cancer, thyroid cancer, endometrial cancer, omentalcancer, testicular cancer, and liver cancer.
 33. The method of claim 20wherein the cancer is lung cancer.
 34. The method of claim 20 whereinthe patient is a human.
 35. The method of claim 20 wherein theexpression vector further comprises a pharmaceutically acceptablecarrier.
 36. The method of claim 35 wherein the expression vector isadministered orally, enterically, intravenously, peritoneally,subcutaneously, transdermally, parenterally, intratumorally, orrectally.
 37. A method for the treatment of cancer comprisingadministering to a patient a therapeutically effective amount of anexpression vector encoding an antisense oligonucleotide that binds to abone morphogenetic protein-4 nucleic acid sequence.
 38. The method ofclaim 37 wherein the expression vector further comprises a selectivepromoter.
 39. The method of claim 38 wherein the expression vector iscarcinoembryonic antigen (CEA) promoter.
 40. The method of claim 37wherein the cancer is a carcinoma.
 41. The method of claim 37 whereinthe carcinoma is selected from the group consisting of bladder cancer,breast cancer, colon cancer, kidney cancer, lung cancer, ovarian cancer,thyroid cancer, endometrial cancer, omental cancer, testicular cancer,and liver cancer.
 42. The method of claim 41 wherein the cancer is lungcancer.
 43. The method of claim 37 wherein the patient is a human. 44.The method of claim 37 wherein the expression vector further comprises apharmaceutically acceptable carrier.
 45. The method of claim 44 whereinthe expression vector is administered orally, enterically,intravenously, peritoneally, subcutaneously, transderinally,parenterally, intratumorally, or rectally.
 46. An article of manufacturecomprising packaging material and, contained within the packagingmaterial, a compound that is a bone morphogenetic protein-4 activityinhibitor, wherein the packaging material indicates that the compoundcan be used for treating cancer in a patient.
 47. The article ofmanufacture of claim 46 wherein the cancer is a carcinoma.
 48. Thearticle of manufacture of claim 47 wherein the carcinoma is selectedfrom the group consisting of bladder cancer, breast cancer, coloncancer, kidney cancer, lung cancer, ovarian cancer, thyroid cancer,endometrial cancer, omental cancer, testicular cancer, and liver cancer.49. The article of manufacture of claim 46 wherein the cancer is lungcancer.
 50. A method for the diagnosis of cancer in a patient,comprising obtaining a biological sample from a patient and measuringthe level of bone morphogenetic protein-4 in the biological sample,wherein an elevated level of bone morphogenetic protein-4 indicatescancer in the patient.
 51. The method of claim 50 wherein the cancer isa carcinoma.
 52. The method of claim 51 wherein the carcinoma isselected from the group consisting of bladder cancer, breast cancer,colon cancer, kidney cancer, lung cancer, ovarian cancer, thyroidcancer, endometrial cancer, omental cancer, testicular cancer, and livercancer.
 53. The method of claim 50, wherein the cancer is lung cancer.54. The method of claim 50 wherein the level of bone morphogeneticprotein-4 is measured by an immunoassay.
 55. The method of claim 54wherein the immunoassay is selected from the group consisting of EnzymeLinked immunosorbent Assay (ELISA), Western blot, immunoprecipitation,in situ immunohistochemistry, and immunofluorescence.
 56. The method ofclaim 50 wherein the assay used to measure the level of bonemorphogenetic protein-4 is Enzyme-Linked Immunosorbent Assay (ELISA).57. The method of claim 50, wherein the biological sample is selectedfrom a group consisting of blood, blood serum, urine, sputum, synovialfluid, ascites, and tissue.
 58. The method of claim 50 wherein thebiological sample is blood serum.
 59. A method for the diagnosis ofcancer in a patient, which method comprises detecting the overexpressionof bone morphogenetic protein-4 in the patient, the overexpression ofbone morphogenetic protein-4 indicating the presence of cancer, themethod comprising the steps of: (i) quantifying in vivo or in vitro thepresence of bone morphogenetic protein-2 in a patient or a biologicalsample obtained from a patient; (ii) comparing the result obtained instep (i) to that of a normal, non-cancerous patient; and (iii)diagnosing for the presence of cancer based on an increased level ofbone morphogenetic protein-4 in step (ii) relative to a normal,non-cancerous patient.
 60. The method of claim 59 wherein the cancer isa carcinoma.
 61. Me method of claim 60 wherein the carcinoma is selectedfrom the group consisting of bladder cancer, breast cancer, coloncancer, kidney cancer, lung cancer, ovarian cancer, thyroid cancer,endometrial cancer, omental cancer, testicular cancer, and liver cancer.62. The method of claim 59 wherein the cancer is lung cancer.
 63. Themethod of claim 59 wherein bone morphogenetic protein-4 is quantified byan immunoassay.
 64. The method of claim 59 wherein the bonemorphogenetic protein-4 is quantified by Enzyme-Linked ImmunosorbentAssay (ELISA).